Fillion was born in Edmonton, Alberta, Canada, the son of Cookie and Bob Fillion, both of whom are retired English teachers. He has an older brother, Jeff, and attended Concordia University College of Alberta and the University of Alberta.

After working in several theatre, television and film productions, including Theatresports with Rapid Fire Theatre and the improvised soap opera Die-Nasty, Fillion moved to New York City in 1994 where he acted in the soap opera One Life to Live as Joey Buchanan, for which he was nominated in 1996 for a Daytime Emmy Award in the "Outstanding Younger Actor" category. In 1997, he left the series to pursue other projects (but would return for a brief guest appearance in 2007). After moving to Los Angeles, he played a supporting role in the sitcom Two Guys, a Girl, and a Pizza Place and was cast as "James Frederick Ryan" or "The Minnesota Ryan" in Steven Spielberg's Saving Private Ryan.

In 2002, Fillion starred as Captain Malcolm Reynolds in the Joss Whedon science fiction television series Firefly, for which he won the "Cinescape Genre Face of the Future - Male" award by the Academy of Science Fiction, Fantasy & Horror Films, USA. Fillion also won the SyFy Genre Awards in 2006 for Best Actor/Television and was runner-up for Best Actor/Movie. Although the show was cancelled, it was adapted to the big screen; Fillion reprised his role as Mal in Whedon's movie Serenity (2005). He also had a recurring role as Caleb in the final season of Whedon's series Buffy The Vampire Slayer. Whedon has a history of reusing actors, and had also used Firefly alumni Gina Torres, Summer Glau and Adam Baldwin on his other show, Angel.

Fillion has lent his voice to the animated series King of the Hill in 2001, the video game Jade Empire (as the voice of Gao the Lesser), and the animated series Justice League Unlimited (as Vigilante in the episodes "Hunter's Moon" and "Patriot Act") in 2005 and 2006. Fillion starred in James Gunn's 2006 horror film Slither. For his starring role as Bill Pardy, he garnered a 2006 Fangoria Chainsaw Awards nomination in the category of Dude You Don't Wanna Mess With. Fillion starred in the romantic comedy film Waitress, written and directed by the late Adrienne Shelly, which premiered at the Sundance Film Festival January 21, 2007 and opened in theaters on May 2, 2007. Waitress Worldwide Box Office Gross as of 9/13/07 $20,063,714 according to Box Office Mojo. He also starred in White Noise 2: The Light. He has made one appearance in the 2006-2007 season of the television show Lost, as Kevin, Kate's ex-husband.

In October 2006, Fillion signed a talent holding contract with the Fox Broadcasting Company, and in December 2006, The Hollywood Reporter confirmed that Fillion was cast in the lead role of Alex Tully in the series Drive, which debuted on FOX in the spring of 2007. Drive was created by Fillion's longtime friend and former Angel and Firefly writer Tim Minear. Ivan Sergei played Alex Tully in the original pilot episode of Drive. The first two Drive episodes premiered on April 13, 2007 in Canada (April 15, 2007 in the United States). However, the show did not deliver the ratings FOX desired, and on April 25, 2007, the network announced that the series was cancelled. The final two produced episodes were supposed to air back-to-back on FOX on July 4, 2007 starting at 8/7c. This date was later changed to July 13, 2007. The final two episodes did not actually become available until July 15, 2007 when they were posted on Drive's MySpace page. Fillion reprised his 1990s role as One Life to Live's Joey for the series' 10,000th episode, aired 17 August 2007.

Fillion and actress Dana Delany joined the cast of ABC's Desperate Housewives at the beginning of the Fall 2007 season. His first episode aired on September 30, 2007. Currently filming movie Trucker according to Nathan Fillion at the Internet Movie Database. Fillion plays the voice of a Marine Sergeant in the Xbox 360 game Halo 3. He is joined by his Firefly co-stars Adam Baldwin and Alan Tudyk who both voice marines. At one point early in the first mission, he identifies himself as "[Sergeant] Reynolds" over the radio, referring to his characters name from the TV series Firefly. All three actors are given personalities in the game that match those of their characters from Firefly.

• Fillion appears in the Martin Firrell project HERO
• Fillion launched the project Kids Need to Read with friend and author P.J. Haarsma, on Aug. 6th, 2007.