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Mrs. Brisby, a timid widowed field mouse, lives in a cinder block with her children in a field on the Fitzgibbons' farm. She prepares to move her family out of the field as plowing time approaches, but her son Timothy has fallen ill. She visits Mr. Ages, another mouse and friend of her late husband, Jonathan, who diagnoses Timothy with pneumonia and provides her with medicine. Mr. Ages warns her that Timothy must stay inside for at least three weeks or he will die. On her way home she encounters Hubie, Marina, Rocko, Splatter, Petra, Beanie, Timmy, Pikatwo, Bonkers, Fawn, Buck, Stanley and Jeremy, a clumsy but friendly crow. They all narrowly escape from the Fitzgibbons' cat, Dragon.

The next day, Mrs. Brisby discovers that Farmer Fitzgibbons has started plowing early. Although her neighbour Auntie Shrew helps her disable his tractor, Mrs. Brisby knows she must devise another plan. Jeremy takes her, Hubie and his friends to visit the Great Owl, who tells them to visit a group of rats that live beneath a rose bush on the farm and ask for Nicodemus, their wise and mystical leader.

The group enter the rose bush and encounters a tough and aggressive guard rat named Brutus, who chases them away with a battle axe. They encounter Mr. Ages (who now has his leg bandaged up), and are amazed to see the rats' use of electricity and other technology.

The team meet Nicodemus and Justin, the Captain of the Guard, and a ruthless, power-hungry rat named Jenner. From Nicodemus Mrs Brisby learns that many years ago her husband, along with the rats and Mr. Ages, were part of a series of experiments at a place known as NIMH, the National Institute of Mental Health. The experiments boosted their intelligence, enabling them to escape, as well as extending their lifespans. However, they are unable to live only as rats, needing human technology to survive, which they have only accomplished by stealing. The rats have concocted "The Plan", which is to leave the farm and live independently. Nicodemus gives Mrs. Brisby an amulet called "The Stone", that gives magical power when its wearer is courageous.

Because of her husband's relationship with the rats, they agree to help Mrs. Brisby move her home. First they need to drug Dragon to sleep, so that they can complete the move safely. Only Mrs. Brisby can do this, as only mice are small enough to fit through the hole leading into the house; Jonathan was killed by Dragon in a previous attempt, while Mr. Ages broke his leg in another. That night, she puts the drug into the cat's food dish, but the Fitzgibbons' son Billy catches her. Marina tells Hubie and the others to go and move the house while she and Pikatwo help Mrs Brisby

During the rescue, Marina, Pikatwo and Mrs Brisby overhears a telephone conversation between Farmer Fitzgibbons and NIMH and learns that the Institute intends to exterminate the rats the next day. The rescue is successful and they run off to warn Justin and the others.

The rats, Hubie and the rest of his friends are moving the Brisby home, with the children and the bird kids inside, using a rope and pulley system during a thunderstorm. Jenner, who wishes for the rats to remain in the rose bush, sabotages the ropes with his reluctant accomplice Sullivan, causing the assembly to fly apart and kill Nicodemus. Mrs. Brisby, Marina and Pikatwo arrive and try to convince the rats that NIMH is coming and they must leave, but Jenner calls them hysterical liars, attacks Mrs Brisby, and attempts to take the amulet from her neck. Sullivan alerts Justin, who rushes to Mrs. Brisby's aid. After mortally wounding Sullivan, Jenner engages Justin in a sword fight, which ends in Justin stabbing him. Justin then addresses the other rats to prepare for their departure from the farm. Jenner recovers and advances on an unaware Justin, but the dying Sullivan hurls his own dagger into Jenner's back, killing him. Pikatwo rushes to Sullivan's aid. In his last breath, Sullivan thanks Pikatwo and his friends for the warning and saving his family.

The Brisby house begins to sink into the mud, but Justin and the rats are unable to raise it. Mrs. Brisby's will to save her children gives power to the amulet, which she uses to lift the house and move it to safety, leading to her also saving Petra, Beanie and Timmy.

The rats depart to Thorn Valley with Justin as their new leader, and Timothy begins to recover. Jeremy eventually meets "Miss Right", another crow who is just as clumsy as he is, and the two fly away together.

Hubie, Marina and their friends say goodbye to Mrs Brisby, who thanks them for everything they've done and they continue on another adventure.

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