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Margalo returns to see Stuart but will be in for a big surprise.
- Geena Davis as Eleanor Little, Stuart, Martha, and George's mother.
- Hugh Laurie as Frederick Little, Stuart, Martha, and George's father and Eleanor's husband.
- Jonathan Lipnicki as George Little, Stuart's “older” brother.
- Marc John Jefferies as Will Powell, George's best friend.
- Will Smith as Mr. Powell, Will's father.
- Amelia Marshall as Rita Powell, Will's mother.
- Michael J. Fox as Stuart Little, a white mouse.
- Melanie Griffith as Margalo, a female yellow bird who is Stuart's best friend.
- Nathan Lane as Snowbell, the family cat who is another one of Stuart's best friends.
- Steve Zahn as Monty, the cat who is another one of Stuart, Margalo and Snowbell's best freinds.
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