In New York City, there is a small house, home to a nice family known as the Littles. One day, Frederick and Eleanor, the parents, go to an orphanage to find a brother for their son, George. While there, they meet Stuart, a small, but charming mouse, who apparently, is human civilized. They adopt him. 

George accepts Stuart as his brother, and he loves him. But there is a problem with Snowbell, the Little family cat.

Snowbell tries to kill Stuart, who narrowly escapes by fleeing into the basement.

Snowbell, however, isn't finished. He and his friend Monty go to an alley that night to visit Smokey, who is the leader of the alley cats. Snowbell doesn't want Stuart killed, but he makes a plan with the alley cats to remove Stuart from the Little house. 

On the day of the 92nd annual Central Park boat race, the Wasp, George’s boat, is finished, and they arrive at Central Park. Anton, another kid, cheats during the race and tries to sink them. But Stuart manages to bite into the line of his sail, rendering Anton's boat inoperable. Stuart subsequently wins the race.

During the celebration that night, the Littles are visited by Reginald and Camille Stout, a mouse couple claiming to be Stuart's parents. After discussion, Stuart reluctantly leaves with his real parents, and they go to their home, a small toy castle on a golf course. 

Three days later, the Littles are visited by Mrs. Keeper, whom they had asked to do a little background research on Stuart. She tells them that, according to the records Stuart's parents died several years ago after a pile of canned food fell on them. The Little realise that Stuart had been kidnapped and call the police.

The Littles decide to place "missing" posters around the city to get help in finding Stuart. 

While going through Central Park, Stuart is ambushed by Smokey and a few cats. He manages to evade them by driving his car into the sewer, but at a cost: he loses the car and his luggage while escaping. Eventually, Stuart finds his way home just as the Littles leave to hang the posters. 

Stuart finds Snowbell at home, and he tells Stuart a lie about how the Littles are very happy without him.

Stuart leaves again. However, Snowbell sees the pain Stuart's absence has caused and realises his selfishness.

Discovering Stuart's location from Monty and the other cats, who intend to eat Stuart, Snowbell goes to Central Park and finds him sitting alone in a bird's nest. Snowbell, however, turns on the other cats and escapes with Stuart, admitting his lie to him and that the family actually does love him.

Then, Smokey orders the cats to kill both Stuart and Snowbell. Stuart responds by taking off Snowbell's collar and using it to lure the cats.

The cats give chase, and eventually corner Stuart hanging from a tree branch.

Smokey and the other cats try to catch Stuart on a lower branch, but Snowbell breaks it at the last minute and sends them into the water below. Smokey sneaks up behind Snowbell and is about to kill him when Stuart releases a thin branch that hits Smokey in the face and knocks him into the water. Enraged and humiliated, Smokey walks off. Monty and the other cats also climb out of the water, whimpering and embarrassed. 

Snowbell and Stuart walk home and Stuart finally meets his family, telling them that Snowbell helped him home. The Littles bring Stuart and Snowbell inside, and close the windows, ready for bed.