An old rat writes in a journal about his friend Jonathan Brisby, who has been killed that day while helping with "the plan". As he holds onto an amulet, he wonders to himself how to help Jonathan's widow, who knows nothing of her husband's association with this rat, or their experience of "NIMH" four years earlier. Deciding to bide his time, he puts the amulet away and says goodbye to his friend.

Elsewhere, Mrs. Brisby, a shy and timid field mouse, lives in a cinder block with her children on the Fitzgibbon's farm. She is preparing to move her family out of the field they live in as plowing time approaches; however, her son Timothy has fallen ill. She visits Mr. Ages, another mouse and old friend of her late husband, who diagnoses her son with pneumonia, and provides her with some medicine from his strange laboratory. Ages warns her that Timothy cannot go outside for at least three weeks or he will die. However, spring plowing begins the next day, and though Mrs. Brisby is able to stop the tractor, she knows she must come up with another plan. With the help of Jeremy, a clumsy but compassionate crow, she visits The Great Owl, a wise creature living in the nearby woods, to ask for help. He initially advises her to move her family regardless of risk, but after discovering she is Jonathan Brisby's widow, he tells her to visit a mysterious group of rats who live beneath a rose bush on the farm and ask for Nicodemus.

Upon visiting the rose bush, Mrs. Brisby runs into Brutus, a young, muscular rat that tries to kill her with his electric spear. She then sees Mr. Ages, who has somehow broken his leg. When she tries to tell him about Brutus, he replies "Oh never mind Brutus". Ages then tries at first to send her away, but is amazed when she tells him about the Great Owl's advice, because as far as he knows "No one has ever seen the Owl and lived to tell about it." Deciding to vouch for her with the rats, Ages leads her to their home, where she is amazed to see their use of electricity and other human technology. She meets Nicodemus, the wise and mystical leader of the rats (who was the journal writer in the beginning), and Justin, an extremely kind and friendly rat who immediately becomes attached to Mrs. Brisby. She learns that her late husband, along with the rats, was a part of a series of experiments at a place known as N.I.M.H. (which stands for the National Institute of Mental Health). Nicodemus also presents Mrs. Brisby with the amulet, which he tells her to keep secret.

Injections performed on the mice and rats boosted their intelligence, one of them being steroids, allowing them to learn to read and to understand things such as complex mechanics and electricity. The experiments also prolonged their lifespan, which is why Jonathan never told his wife, as she would have aged and died far faster than he. The rats, along with Mr. Ages and Jonathan Brisby, escaped from N.I.M.H. and came to live on the Fitzgibbon farm. The rats created a home for themselves under Mrs. Fitzgibbon's rose bush, creating an elaborate habitation of beautiful chambers, elevators, and Christmas lights. However, the rats are unhappy (on a number of levels) in their dependence on the humans, whom they are stealing electricity from, and have concocted "The Plan", which is to leave the farm and live independently. Because of her husband's prior relationship with the rats, they agree to help Mrs. Brisby move her home out of the path of the plow. However, Jenner and his unwilling accomplice Sullivan, who wish to remain beneath the rose bush, plot to kill Nicodemus during the move. Mrs. Brisby is told by Justin that someone must drug the Fitzgibbon's cat, Dragon, so that they can complete the move safely. But only mice are small enough to fit through the mousehole leading to the house, and Jonathan was killed by Dragon while trying. Mrs. Brisby volunteers as a means of repaying them.

On her way home, Mrs. Brisby sees Jeremy again. Jeremy is dazzled by her amulet and begs her to trade it to him, almost to the point of obsession, but she knows she must keep it and distracts him by asking him to fetch string for her to help move her house. Jeremy heads away to do so.

Later that night, she successfully puts the drug into the cat's food dish, however the Fitzgibbon's son Billy catches her and convinces his mother to let him keep her as a pet. While trapped in a birdcage, she overhears a telephone conversation between Mr. Fitzgibbon and NIMH and learns that NIMH intends to come to the farm to exterminate the rats the next day. She manages to escape from the cage and runs off to warn Justin.

Meanwhile, the rats are completing the move during a thunderstorm. Just as the Brisby house is over Nicodemus, Jenner cuts the pulley ropes, expecting the house to fall and crush Nicodemus. However, Sullivan reneges on his part of the plan, causing the house to land elsewhere. Despite this, the now unsecured equipment is destroyed by the wild weather and Nicodemus is instead crushed under flying pulleys and ropes. Everyone still assumes that it was an accident and Jenner tries to assert himself as the new leader, trying to convince them to return to the rosebush and abandon the plans to migrate. Mrs. Brisby arrives and, though saddened to hear of Nicodemus' death and worried for her children, tries equally hard to convince the rats that NIMH is coming and that they must leave immediately. Jenner becomes angry and knocks her down, allowing him to see the amulet. Overcome by a lust for power, he draws his sword and tries to take it. Justin rushes to Mrs. Brisby's aid, though he is initially unarmed. Jenner stabs Sullivan after Sullivan manages to give a sword to Justin. During the fight Justin accuses Jenner of killing Nicodemus and Jenner admits to it, telling Justin that Nicodemus wanted to ruin everything. Jenner appears to have the upper hand but Justin turns the tables in his favour and stabs him in the stomache. Justin assumes leadership and tells everyone present that they must move tonight. A furious Jenner attacks Justin, however, shortly before dying, Sullivan manages to toss a dagger into Jenner's back, killing him.

Mrs. Brisby suddenly hears the cries of her children from inside the house and realizes that the house is sinking in the mud it landed in. Despite the best efforts of the rats, they are unable to pull it from the mud. However, Mrs. Brisby's will to save her children somehow gives power to the amulet, which she uses to lift the house out of the mud and move it to safety from the plow. The considerable effort causes her to pass out shortly afterwards.

Some time later, Timothy has begun to recover. Jeremy comes with the string, but despairs when he sees the house is already moved. Suddenly finding "Miss Right", an equally clumsy crow who crashes into him, he once again begs Mrs. Brisby for the amulet. She reveals the rats moved from the farm and she gave the amulet to Justin, now the leader of the rats. Jeremy is disappointed but quickly realizes it doesn't matter and he flies off with his new love.

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