Eleanor Little (Geena Davis) and Frederick Little (Hugh Laurie) go to the orphanage to adopt a younger brother for their son George (Jonathan Lipnicki). At the orphanage, they meet and fall in love with an anthropomorphic mouse named Stuart (voiced by Michael J. Fox). Despite discouragement from Mrs. Keeper (Julia Sweeney), they adopt Stuart and take him home. However, Stuart is poorly received by George, who refuses to acknowledge the mouse as his brother, and the family cat Snowbell (Nathan Lane), who is against the idea of having a mouse for a “master”.
Stuart’s new life at the house gets off to a bad start when George unknowingly traps him in a pile of laundry and his mother puts the laundry in the washing machine. Stuart quickly feels like ostracised in the large Little family, especially when George declares Stuart is not his brother, but simply a mouse. When Stuart tells his parents that he is lonely, they ask Mrs. Keeper to do some background research on Stuart’s biological family. After accidentally stumbling across George’s playroom in the basement one day, Stuart and George get to know each other and plan to finish George’s remote-controlled boat, the Wasp, for an upcoming boat race in Central Park. At the same time, one of Snowbell’s alley cat friends, Monty (Steve Zahn), visits unexpectedly and discovers Stuart. Determined not to have his reputation ruined, Snowbell meets with Monty’s leader, Smokey (Chazz Palminteri), a mafia don-like alley cat, and plots to remove Stuart from the household without harming him.
Stuart and George finish the Wasp in time, but when the controller gets broken on the day of the race, Stuart pilots the Wasp himself. He ends up entangled with a larger boat belonging to George’s rival, Anton (Miles Marsico) who always cheats by crashing other competitors boats, but Stuart snaps the wires of Anton’s boat, making it inoperable, and manages to win the race. After this, George feels more comfortable with Stuart and calls him “his brother”. During the family celebration, however, the Littles are visited by a mouse couple, Reginald and Camille Stout (Bruno Kirby and Jennifer Tilly), who claim to be Stuart’s parents and that they gave him up for adoption years ago due to poverty. Somberly, Stuart leaves with the Stouts. Three days later, Mrs. Keeper comes to visit and tells the Littles that Stuart’s parents actually died many years ago in a supermarket accident. Realizing their son has been kidnapped, the Littles call the police, who start a search operation.
That night, Snowbell meets with Smokey and the alley cats, who reveal they had forced the Stouts to pose as Stuart’s parents in order to remove Stuart from the household. Fearing retribution should the Littles discover Snowbell’s deception, Smokey orders the Stouts to hand Stuart over to them. However, the Stouts, having bonded with Stuart, tell him the truth and instruct him to escape. Smokey subsequently orders a mouse hunt for Stuart. He and the alley cats confront him in Central Park, and chase him into a sewer drain, but he manages to outrun them, losing his roadster in the process. He returns home, but finds the Littles absent, going out to put up posters of him. The only one present is Snowbell, who lies that the Littles have been enjoying themselves since Stuart’s departure and uses Stuart’s removed face from the family photograph as proof (which they had actually used for the posters). Crestfallen, Stuart leaves again.
When the Littles return with no success of finding Stuart, Snowbell starts to recognize his selfishness and feels guilty for what he has done. The cats pinpoint Stuart’s location in Central Park and bring Snowbell along for the hunt. However, he finds Stuart thoughtfully sitting in a bird’s nest in a tree and decides to save him from the cats, admitting that he lied and the Littles do love him. Snowbell then tries to reason with Smokey to leave Stuart alone, but Smokey refuses to listen and demands his gang to kill both Stuart and Snowbell. Stuart lures the cats away, but is left hanging from a tree branch. Monty and the cats group together on a lower branch to catch Stuart, but Snowbell breaks the branch they are standing on, sending them falling down into the river below. Smokey, seeking revenge, sneaks up from behind Snowbell and is about to kill Snowbell, but Stuart releases a thin branch that hits Smokey in the face, and sends him falling into the water below. Devastated, Smokey runs off, only to be chased away by a pack of vicious dogs. Stuart rides Snowbell all the way home and they share a happy reunion with the Little family when they arrive to find Stuart safely back home.