The film was followed by the third and final film a direct-to-video sequel, entitled Stuart Little 3: Call of the Wild in 2006.
Three years after the first film, Stuart Little questions his ability after a grueling soccer match alongside George, who kicked him with a soccer ball. He becomes even more downhearted after George's toy airplane gets broken in an accident because of him. However, Stuart's father, Frederick Little, tells him that for every Little, there is a "silver lining", a good thing that comes out of an apparently bad situation.
On his way home from school, Stuart saves a female canary named Margalo who is being pursued by a peregrine falcon, and they become friends. But she is secretly working with Falcon to case and steal from households. When he presses her to find and take an object of value, or lose the sanctuary he promised her, she can't seem to concentrate on her assignment, as she is beginning to fall in love with Stuart. Falcon eventually loses patience and threatens to kill him if she doesn't deliver. Worried for his safety, she takes Eleanor Little's diamond wedding ring.
When the Littles see that the ring is missing, they think it has fallen down the sink. Stuart offers to be lowered down the drain on a string to get it, and nearly succeeds. When the string breaks Margalo saves him, and his thanks to her only makes her feel even more guilty, so she decides to leave. When he can't find her, he assumes she has been kidnapped - and that Falcon is somehow involved. He leaves on a quest to rescue her with the household's reluctant cat Snowbell, but not before setting up a plan with George.
Stuart and Snowbell enlist the help of Monty, who tells them that Falcon's lair is at the disused observation deck of the nearby Pishkin Building. They use balloons to get Stuart to the top, where he finds out that Margalo is Falcon's slave, and was forced to take the ring. He tries to save her, but Falcon captures him, and drops him in a garbage truck. Falcon then shuts Margalo inside a paint can as punishment for rebelling against him. Meanwhile, Snowbell makes his way to the top of the building while the Falcon is absent and frees Margalo, who tearfully tells Snowbell that Falcon killed Stuart. Distraught, Snowbell vows revenge.
On a garbage scow where he has ended up, Stuart blames himself for everything, and has almost lost all hope. Suddenly, he finds George's broken plane, fixes it up, and flies to save Margalo. Falcon returns and almost hits Snowbell off the building, but Margalo defies him by taking the ring and fleeing. Falcon gives chase, but Stuart catches up in the plane and saves Margalo while trying to evade the Falcon. The Littles, who have discovered his absence and whereabouts follow him by taxi as he begins an aerial adventure through the park, with Margalo at his side. They lose Falcon, but he catches up and makes an attempt to kill Stuart, when he detaches the plane's upper wing, damaging the main one and causing it to enter a steep nose dive, which fails when Stuart recovers from the dive, nearly missing the Littles. Unable to run from Falcon, he lets Margalo off. He turns and flies the damaged plane in a kamikaze run while Falcon goes into an attack dive. He uses Mrs. Little's ring to temporarily blind him, and jumps out using a bandana as a parachute. The kamikaze attack works and Falcon is struck head on and defeated. Although he survives the attack, he falls out of the sky and lands in a garbage can that Monty is scavenging in, and is presumably eaten by him, but not before Stuart falls when his parachute is sliced apart by the propeller of the shattered plane, and then is rescued by Margalo.
Stuart is congratulated by his family, and Margalo, who gives Mrs. Little her ring back, and Snowbell reunites with them as well. Soon after, Margalo leaves with the other birds to migrate south, but not before saying goodbye to her friends. Stuart says the "silver lining" is that she'll be back in the spring, and his baby sister, Martha, says her first words: "Bye bye, birdie.", which the family then celebrates and then head inside to the comfort of their home.
- Michael J. Fox (voice) as Stuart Little
- Melanie Griffith (voice) as Margalo
- Nathan Lane (voice) as Snowbell, the Little family cat.
- Geena Davis as Eleanor Little
- Hugh Laurie as Frederick Little
- Jonathan Lipnicki as George Little
- James Woods (voice) as Falcon
- Steve Zahn (voice) as Monty, Snowbell's friend.
- Anna and Ashley Hoelck as Martha Little
- Marc John Jefferies as Will, George's friend
- Jim Doughan (who voiced Lucky in the first film) as Stuart and George's soccer coach