Roughly eight months after River and Simon have joined Serenity's crew, the ship's captain Malcolm "Mal" Reynolds overrides Simon's objections and insists upon bringing River and her talents along on a bank raid. The raid is interrupted by a Reaver attack. After the raid-team's narrow escape back to Serenity, Simon confronts Mal, who decides to leave the two fugitives behind at their next stop. At said stop, River starts a deadly commotion that is triggered by a subliminal message broadcast by the Alliance in a commercial. Mal allows Simon and River to continue traveling on Serenity while the crew contacts a reclusive techno-geek known as Mr. Universe. Mr. Universe discovers the subliminal message and also notes that River whispered the name "Miranda." Mal receives a call from Inara, a former passenger. Suspecting a trap, but mindful that she must be in danger, Mal visits her and is confronted by the Operative, who offers to let Mal go if he turns River over to him. Mal refuses, and thanks to Inara's quick thinking, they escape from the Operative and back to Serenity. Another of River's outbursts shows the crew that "Miranda" is an unknown outer rim planet, about which River learned something horrible when in contact with members of Parliament during an inspection visit of her training. Since traveling to Miranda would require a suicidal crossing into Reaver territory, Serenity instead goes to Haven, now home to another former passenger, Shepherd Book. On arrival, the crew discovers that the outpost has been ravaged by Alliance forces, fatally wounding Shepherd Book in the process. The Operative sends a message claiming responsibility, and promising more of the same until River is turned over. Again overriding the crew's complaints, Mal orders that Serenity be made to look like a Reaver ship. They successfully sneak the disguised vessel through an entire fleet of Reaver warships. Upon reaching Miranda, the crew finds it has a completely habitable environment, but the sprawling cities are empty except for masses of badly decomposed corpses, all without apparent cause of death. The crew discovers a log recorded after the disaster by an Alliance expedition, which reveals that the Alliance administered a chemical substance designed to suppress aggression and thus render the planet free of violence. It worked so well that the populace stopped working, eating, or caring about anything and let themselves die. However, a tenth of a percent of the population had the opposite reaction, turning hyper-aggressive and mentally unstable: they became the Reavers. A sickened Mal plans to reveal this secret to all the worlds by using Mr. Universe's transmitter equipment, but the Operative has anticipated this and waits just outside of Reaver space with an Alliance fleet. During the return trip through the Reavers, Serenity opens fire upon a pursuing ship, causing the other vessels to chase it: Serenity emerges from Reaver space flanked by the entire Reaver fleet. A massive battle ensues between the Alliance and the Reavers, allowing Serenity's pilot Wash to fly down to Mr. Universe's planet. The Operative's ship is destroyed, and he also flees to the surface in an escape pod. Serenity crash lands on the planet and while it suffers massive damage, the crew has survived. Just as everyone begins to relax, a Reaver harpoon impales Wash, killing him instantly. Fleeing Serenity, the crew sets up a last stand to give Mal the time he needs to get to Mr. Universe and transmit the message. Mal finds Mr. Universe murdered, but triggers a message revealing a hidden backup transmitter. The Reavers attack the crew, inflict several injuries and force them to retreat. The crew tries to close a blast door, but it will not seal completely, so River dives through the gap in the door and seals it from the other side trapping herself with the Reavers. Mal reaches the secondary transmitter, but then The Operative appears, leading to a final bloody showdown between the two men. Mal emerges as the victor but does not kill the Operative, instead leaving him to watch the recording from Miranda. Mal returns to his crew; just as he is told what River did, the blast door opens to reveal River standing unscathed amidst an enormous pile of dead Reavers. A squad of Alliance troops belatedly storm onto the scene, but the Operative, his faith in his mission shattered by the Miranda message, orders them to stand down. The crew buries Mr. Universe, Shepherd Book, and Wash and patches up Serenity. As they prepare to resume their travels, the Operative makes his own exit, promising Mal they will never meet again. Serenity blasts off with Mal in Wash's seat at the helm, and River acting as his copilot.

However, some reviewers felt the film was unable to overcome its television origins, and did not successfully accomplish the transition to the big screen. USA Today wrote that "the characters are generally uninteresting and one-dimensional, and the futuristic Western-style plot grows tedious" while Variety declared that the film "bounces around to sometimes memorable effect but rarely soars".

Despite critical acclaim and Internet buzz, Serenity performed modestly at best at the box office. Although several pundits predicted a #1 opening, the film opened at #2 in the United States, taking in $10.1 million its first weekend, spending two weeks in the top ten, and totaling a box office gross of $25.5 million. Movie industry analyst Brandon Gray described Serenity's box office performance as "like a below average genre picture".

Serenity's international box office results were mixed, with strong openings in the UK, Portugal and Russia, but poor results in Spain, Australia, France and Italy. Universal International Pictures canceled the film's theatrical release in at least seven countries, planning to release it directly to DVD instead. The box office income outside the United States was $13.3 million, with a worldwide total of $38.8 million, slightly less than the film's $39 million budget, which does not include the promotion and advertising costs.

The Operative embodies the Alliance and is, as Whedon described, the "perfect product of what's wrong with the Alliance". He is someone whose motives are to achieve a good end, a "world without sin". The Operative believes so strongly in this idea that he willingly compromises his humanity in furtherance of it. In contrast, Mal is, at the movie's beginning, a man who has lost all faith. By the end of the movie, however, Mal has finally come to believe in something strongly that he becomes willing to lay down his life to preserve it. Whedon has said that the most important line in the film is Mal's contented promise to the Operative at its climax: "I'm going to show you a world without sin." Whedon's point is that a world without sin is a world without choice, and that choice is ultimately what defines humanity.

Joss Whedon explains in the DVD commentary track that the planet "Miranda" received its name in reference to a line spoken by William Shakespeare's Miranda in The Tempest, Act V, scene I: "O brave new world, / That has such people in't!" The Alliance had hoped that Miranda would be a new kind of world, filled with peaceful, happy people, and represents the "inane optimism of the Alliance". It is also of note that the planet on which River was conditioned is called Ariel, which is also the name of a character in The Tempest. (Significantly, in The Tempest, the spirit Ariel acts under an obligation to the father of Miranda until freed at the play's conclusion.) The aforementioned quotation from The Tempest also gave the title to the novel Brave New World by Aldous Huxley, which portrays an orderly but passionless society in which the populace are kept in check by means of a drug, Soma. Huxley described his novel as a "negative utopia". Like the The World State of Brave New World, the Alliance in Serenity attempts to create a perfect society by administering a drug, but their manipulation leads to the exact opposite.

Serenity also owes a debt to such science fiction classics that influenced it such as Blade Runner, Star Wars and Aliens.

The Fruity Oaty Bar commercial is partially inspired by Mr. Sparkle, the mascot of a fictional brand of dish-washing detergent, who was featured in The Simpsons episode "In Marge We Trust". Serenity is one of several films set in the future that speculates how popular culture might evolve. Other films include Blade Runner, A Clockwork Orange and The Fifth Element. This future envisioned in Serenity has two political and cultural centers: Euro-American and Chinese. Characters all speak English and Mandarin, with the latter language reserved for the strongest curse words. However, the tones and pronunciation of these Mandarin words are barely recognizable to the native Chinese speaker. While these two are the dominant languages of the film, other languages are also spoken in the Firefly / Serenity universe, including Russian (spoken by Simon during the movie). The safeword phrase that Simon uses to shut River down, "Eta kuram na smekh", is a Russian expression. Literally, it means, "That's for chickens to laugh at", a Russian idiom for "That's ridiculous." The English subtitles on the DVD incorrectly list the line as "[speaking Chinese]".

There is a Biblical reference after Mal tells Mr. Universe about the Miranda tape. Mr. Universe asks The Operative (who was standing right behind him while he was talking to Mal) for his "thirty coin", referring to the thirty pieces of silver that Judas was paid to betray Jesus.

The criminals 'Fanty' and 'Mingo' whom we meet early in the movie (when River is mesmerized by the television ad in the bar) are probably references to a pair of criminals of the same names ('Fante' and 'Mingo') in the film noir The Big Combo.

Whedon's directions to Newman for the Serenity theme were that he wanted something homemade and mournful that would let viewers know that they were now "home" and evoke the idea of the pioneer, when everyone only had what they could carry.

On June 23, 2006 a number of fans organized and spread word of "Serenity Day," on which all fans were proposed to purchase a copy of Serenity in an attempt to convince Universal that a sequel would be profitable. The significance of this day is that June 23, 2006, is the one-year anniversary of the third and final advance screening of Serenity prior to its release, and also Joss Whedon's birthday. The impact of the event could be seen from Serenity reaching #2 in the Amazon DVD Charts.

On October 1, 2006, Whedon posted a comment to the Whedonesque.com website, responding to a rumor that he was currently working on a sequel to Serenity. He wrote, "There's no sequel, no secret project regarding Serenity or some such and I'm not even sure how anyone thought there was talk there. I've seen Nathan and Tim (and Summer and Alan) recently because they're my friends because I'm so, yeah, awesome. So let's put that to bed and smother it with a pillow."

Whedon's response to the rumor consequently sparked many websites to publish articles stating that he would never work on a sequel to Serenity. Joss again returned to Whedonesque.com to respond to the new stories and wrote, "Holy Mother of Oats! I turn my back for five minutes (that's how long it takes to admire my lovely back) and the interweb goes banoonoos! Isn't there any ACTUAL news to get wrong? Sorry about all this; it might be best if I just stay off the computer for a while....Here's a thing: when Firefly was canceled, my heart got broke. Sounds a bit much, but it changed me. Not even Serenity could patch that wound. I'm wearier, warier -- after all those years as a movie writer, you'd think I'd be prepared for another lesson on my unimportance in the scheme of things, but I wasn't....All these rumor of projects or the death of projects... When the two worlds align and something actually happens, whatever it is, you guys know I'll be on this site as soon as I'm allowed to be. And I'll be very very clear. There is no news. Not never, just now."

Bridging the gap between the end of the television series and the beginning of the movie is a three-issue comic book series entitled Serenity: Those Left Behind. The comic is written by Joss Whedon and Brett Matthews, illustrated by Will Conrad and Laura Martin, and published by Dark Horse Comics. The story focuses on the crew of Serenity taking a salvage job from Badger following a botched theft on a backwater planet, and the pursuit of River by the ominous blue-gloved men seen in the television series. The story is considered part of the Firefly/Serenity canon. Each issue of the series features three different covers, with each cover featuring one of the nine main characters, each by a different illustrator, including Joe Quesada, Bryan Hitch, Tim Bradstreet and John Cassaday. The first issue was published in July 2005, and the final one appeared in September. The comics quickly sold out on release, with both the #1 and #2 issues going into second printings. Many comic book retailers reported that it was their highest selling comic those months, in part because of the multiple covers, but also because it attracted a group of customers who are not traditionally comic book buyers. The comics were later reissued as a trade paperback compilation.

It has been recently confirmed that Joss Whedon will reteam with Brett Matthews to write Serenity: Better Days for Dark Horse comics to be released in the fall of 2007 as we see what happens when a heist goes right and the crew finds themselves in a rare place: on easy street. "This new story will take place in the 'Firefly' years i.e., everyone's alive," teases Whedon. "Basically, they pull off a heist and everything doesn't go completely wrong. This, needless to say, has never happened, and it's about how they deal with success."