Mrs. Brisby, a shy and timid field mouse, lives in a cinder block with her children on the Fitzgibbons' 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 provides her with some medicine from his laboratory. Mr. Ages warns her that Timothy cannot go outside for at least three weeks or he will die. On her way back home she encounters Jeremy, a clumsy but compassionate crow and Winnie the Pooh, Piglet, Tigger, Rabbit, Eeyore, Ash Ketchum, Pikachu, Misty, Togepi, and Brock.
The next day spring plowing begins, and though Mrs. Brisby is able to stop the tractor, she knows she must come up with another plan. With the help of Jeremy she and the others visit the Great Owl, a wise creature living in the nearby woods, to ask for help. He tells them to visit a mysterious group of rats who live beneath a rose bush on the farm and ask for Nicodemus.
Mrs. Brisby and Pooh and the others go into the rats' home, where they are amazed to see their use of electricity and other human technology. They meet Nicodemus, the wise and mystical leader of the rats, and Justin, an extremely kind and friendly rat who is the Captain of the Guards. She and the others learn that her late husband, along with the rats and Mr. Ages, was a part of a series of experiments at a place known as NIMH (which stands for the National Institute of Mental Health). The experiments had boosted their intelligence to human level, allowing them to easily escape. However, the rats have concocted "The Plan", which is to leave the farm and live without stealing electricity from humans. Nicodemus then gives Mrs. Brisby an amulet that gives magical power when its wearer is courageous.
Because of her husband's prior relationship with the rats, they agree to help Mrs. Brisby and the the others move her home out of the path of the plow. Mrs. Brisby volunteers to drug the Fitzgibbon's cat, Dragon, so that they can complete the move safely. Only mice are small enough to fit through the mouse hole leading to the house, and Jonathan was killed by Dragon while trying. Later that night, she successfully puts the drug into the cat's food dish, but 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 and Pooh and the others are completing the move during a thunderstorm. However, Jenner and his hesitant accomplice Sullivan and Jessie, James, and Meowth of Team Rocket, who wish to remain in the rose bush, kill Nicodemus, making it look like an accident. Mrs. Brisby arrives and tries to convince the rats that NIMH is coming and that they must leave immediately. Jenner becomes angry and knocks her down. Team Rocket started to capture Pooh and the others. Justin rushes to their aid, and a sword fight between him and Jenner ensues, which ends with Sullivan, himself mortally wounded, killing Jenner and saving Justin's life. Tigger uses a caterpult to blast off Team Rocket.
Mrs. Brisby suddenly realizes that the house is sinking in the mud it landed in. Despite the best efforts of the rats and the others, they are unable to pull it from the mud. However, Mrs. Brisby's will to save her children 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 next morning, the rats have already gone to Thorn Valley with Justin as their new leader and Timothy has begun to recover. Jeremy also finds "Miss Right", an equally clumsy crow, and the two fly away together and Pooh and the others started to leave to their next adventure.
- Ash Ketchum, Pikachu, Misty, Brock, Togepi, and Team Rocket (Jessie, James, and Meowth) guest star in this movie.
- The storyline continues in Pooh's Adventures of The Secret of NIMH 2: Timmy to the Rescue.
- This is an NTSC film with NTSC bits from The Tigger Movie, Winnie the Pooh: A Very Merry Pooh Year,Pooh's Heffalump Movie, the Pokemon movies 1, 2, 3, and 5 and Pokemon: Mewtwo Returns and PAL bits from The Many Adventures of Winnie the Pooh, Pooh's Grand Adventure: The Search For Christopher Robin,Piglet's Big Movie, Pooh's Heffalump Halloween Movie, and Pokemon 4Ever.
- The Secret of NIMH was first released on DVD in 1999, the same year that Pokemon: The First Movie was released in theaters and Winnie the Pooh: Seasons of Giving was released straight to video.