The movie opens with a little boy running out of his room early in the morning and running to wake his parents yelling “It’s today, ‘it’s today!” His parents prepare and take him to school while he asks them lots of questions and suggests to come with them. He reminds them he wants a little brother and not a big brother right before the school bus takes him away.
His parents, the Littles meet the head of the adoption agency and are allowed to explore the facility and meet the orphaned children. There are so many of them and they sit, overwhelmed at the sight of the kids. They meet a talking mouse who is so happy with the way the husband and wife are so in tune with one another they literally know what the other person is going to say. The Mouse tries to help them choose, he tells them Susan can read French while Edith can tap dance and blow bubbles at the same time. If they wanted a boy, the mouse says Benny can do handstands while Andy can run fast.
The family learns that the mouse has been at the orphanage for way too long which is why he knows so much about everyone. No one wants to adopt a mouse. The littles make their decision, they want the mouse, Stuart. The head of the agency warns them about adopting out of their species and the dangers associated with it but they insist and adopt Stuart. They bring Stuart home and tell him the house can be found easily by every little from any part of the world. Just as they are showing Stuart around, the family cat, Snowbell pounces and grabs Stuart between his teeth. They warn Snowbell never to eat Stuart as he is a member of the family now.
George, the little boy comes home from school, excited to see his new adopted brother. George is disappointed, it wasn’t what he expected to see. Stuart is sad that his new brother doesn’t like him. At the dinner table, George asks Stuart to pass the Gravy, a task impossible for a mouse. The Littles try to make Stuart feel at home and after tucking him in at night, he calls them mom and dad.
Snowbell comes in and jumps on the bed, angry that Stuart has a comfy bed while he sleeps in a rag in the corner. He is angry that Stuart thinks he is his pet. He warns Stuart to stay away from the windows so the other cats in the neighborhood won’t see him as it will ruin his reputation.
In the morning, both George and Stuart go through the same routines, brushing their teeth after their mother, Mrs. Little had woken them. When Mrs. Little asks George to send her his laundry, George mistakenly drops his clothes on Stuart and sends them through the laundry shoot to Mrs. Little who places them right in the washer without knowing. Snowbell wouldn’t help and Stuart swallowed some detergent and nearly drowned in the washer.
George is still unhappy, he doesn’t want to go shopping with his new brother, his father suggests he will like his brother more if he will only spends a little time with him. They talk about the boat racing event coming up, Mr. Little advises him to finish his boat faster so he could participate but George doesn’t want to participate because he always finishes last.
Mrs. Little talks to the tailor about Stuart’s special clothing needs. Stuart tries out his new clothes and is happy to hear he looks just like a member of the Little family. That evening, there is a party to present Stuart to the rest of the large extended family. When he is introduced, the family is surprised but recovers quickly. All the gifts they brought are for a human boy and there is a slight air of sadness that everyone tries to cover up. Stuart makes a moving speech and makes the family happy.
Uncle Crenshaw and Mr. Little hand George a ball which he should enjoy with his new brother. They ask him to go outside and throw the ball around with his new brother, but George asks them how they expect him to throw the ball around with a mouse, or ride a bicycle and do all of the things they want him to do with the little boy. Everyone leaves to avoid the awkwardness. That night, Stuart climbs into the Little bed and asks them about his real family, he says he has an empty space inside of him and asks them about his real family, the ones that look like him. The littles promise to help.
They go to the adoption agency and ask if they could help find Stuart’s real family. The agency say they can’t help. At home, Stuart tries to start over with Snowbell, but Snow doesn’t want to fraternize with a mouse. A neighborhood cat, Monty the mouth tries to get into the house and Snowbell tries to keep him away so he won’t see the mouse. He sneaks in and eats out of Snowbell’s plate. Snowbell tries to hurry the cat so he doesn’t see Stuart as he is afraid the news of the mouse in his home will spread over the neighborhood.
Just as he is leaving, he sees the mouse and comes back in to catch it. He is surprised when Stuart doesn’t run and laughs Snowbell to scorn when he learns that Stuart is a member of the Little family. Snowbell is angry and chases Stuart around. He runs into George’s room. He falls into a western scene George built with his father. George doesn’t want to, but Stuart convinces him to allow him to play with the train set. They have some fun and George lets him ride in his little car, Stuart feels happy and says it’s the first time he’s fit in since he got here. He sees the unfinished boat George and his father were building.
The boat was so fine, and Stuart asks why it isn’t finished. George explains that he doesn’t want to race again as he is afraid he will lose. Stuart asks to help but George says he isn’t sure he wants a brother, Stuart asks what about a friend, and George agrees. Their parents return and are happy to see them bonding, they agree to go to the races together and that comes up in two days’ time.
Snowbell is sad watching the family bond. He reaches out to Monty the mouse and the cat links him up with an alley cat, Smokey is able to handle the Stuart issue for him. Smokey is angry, says a cat can’t have a rodent for a master and agrees to take care of Stuart.
It’s race day, George and Stuart are working on their boat when Anton, a mean kid tells George he will finish last. While Stuart goes to get the remote, George is scared he might lose and wants to pull out of the race. While Stuart is getting the remote, he trips and the remote falls off his hands and someone steps on it. George is sad, Stuart is even sadder that he has ruined their first family outing. The race starts, and the family were talking about going home but Stuart got on the boat, ready to sail it by hand. Mrs. Little wants Stuart out of the water, she is scared but Stuart is hell bent on sailing the boat, he promises he won’t let George down.
Anton is a cheater, he uses his boat to run over other people’s boats just to get ahead. Anton tries to run George’s boat over with Stuart in it and George fights him. Meanwhile, Stuart tries to repair the broken sail. Anton loses control of his boat and Stuart wins the race with George’s boat. When someone asks, Who is that mouse anyway, George was quick to say it’s not a mouse but his brother. At home, the whole family celebrate the victory and take a most beautiful family photograph.
Just as they were at their happiest a mouse couple comes asking after Stuart and says they are his parents. They have a good story for why they let him go. They convince the family to let Stuart go with them and Stuart packs up. George was sad and wouldn’t say goodbye. The littles are sad, and say a teary goodbye to Stuart who is also sad but left with his new found family. Mrs. Little feels something is not right but Mr. Little convinces her to let it go. George offers Stuart the toy car as a parting gift.
Instead of a taxi, Stuart and his new found parents drive off in the small car. The new family take Start to a rough area and tell tales of rough living. Back at the Little household, Mrs. Little is sad and while she is crying, the adoption agency head comes over to give them news of Stuart’s parents, they had died in an accident a long time ago. They are surprised to hear this and Mrs. Little asks her husband to call the police. Just as the police arrive, Snowbell runs out to tell Monty that the jig is up, Smokey proffers the best solution, they have to kill Stuart like they should have done at the beginning.
The police say the kidnappers probably want to kill Stuart since the kidnappers didn’t call for a ransom and if they don’t want money, then they must be looking to do serious harm to Stuart. The Little’s are devastated.
The Stouts, who kidnapped Stuart plan to deliver him into the hands of Smokey so he will be killed. Mrs. Stout was sad, she was crying so hard and forces Mr. Stout to tell him the truth. Stuart was happy to know they are not his real parents, now he can return home to the Littles. He sets off in his car, heading off back to the Little House.
Back home, all the Littles have gathered, George wasn’t going to give up on his brother, he begs his extended family to make posters and post all over town. While they make posters, Stuart is driving home. The cats hear that he had been warned and plan to ambush him at the park. They find him, surround him and he tears off quickly, driving backwards, away from the cats. He falls into the sewer system and the cats can’t get into the sewer system.
The Stuarts divide themselves and leave to cover all parts of town including the park but just as they leave, Stewart gets home to meet an empty house. Only Snowbell is in the house. The cat tries to convince Stuart that the Littles have been celebrating since he left. He didn’t believe at first, but when Snowbell shows him he’s been cut out of the family photos, Stuart falls for it. He becomes sad, cries and leaves the house.
The Littles just finished up at the park and headed home, hopeful of finding Stuart while Stuart is just leaving the Little home, sadly ambling away into the night. George sits by the phone every day, waiting to hear someone call to say they’ve found Stuart but nobody calls. Monty comes to invite Snowbell because the cats saw Stuart going into the Park. Snowbell is the one that finds Stuart in the tree and tries to protect him but the other cats find out and ask Snowbell to bring him down.
Snowbell couldn’t do it, instead, he takes Stuart and tries to escape from the other cats. Snowbell rebels against the other cats and protects Stuart. Stuart and Snowbell team up to destroy the other cats.
After they’ve defeated their adversaries, they head home together. The Littles were about to go to bed when Stuart comes home and the family is reunited again. They were all happy once more.