It's been two years since Frederick (HUGH LAURIE) and Eleanor Little (GEENA DAVIS) adopted Stuart (voice of MICHAEL J. FOX), a diminutive and talking mouse, into their home and family and he's adjusted quite well since then. Now rooming with his older human brother George (JONATHAN LIPNICKI) and getting along with the family's talkative but neurotic Persian cat Snowbell (voice of NATHAN LANE), Stuart has seen the addition of a human baby sister, Martha (ANNA & ASHLEY HOELCK).
Yet, Stuart feels the pangs of growing up. Not only is his mother smothering him with her overprotective worrying, but he finds that others, including George, are too busy to do anything with him. Thus, when Margalo (voice of MELANIE GRIFFITH), a small and soft-spoken bird literally falls from the sky and nearly into his lap, Stuart is happy to have found a kindred spirit and fellow small being.
Unbeknownst to him, however, Margalo is really working for the Falcon (voice of JAMES WOODS), a malevolent bird of prey that's attempting to find what the Littles possess that might be worth stealing. Margalo isn't happy to be under the Falcon's wing, but is coerced into robbing Eleanor's ring.
When she then leaves without saying goodbye, Stuart believes the Falcon has abducted her. With the help of Snowbell and his feline friend, Monty (voice of STEVE ZAHN), and getting George to cover for his absence, Stuart then sets out to find and rescue Margalo, unaware of the adventure and danger that lie in store for him.