- Sean Astin as Space Mouse, a house mouse from Rodentia.
- Jason Biggs as Mr. Squiggles, Space Mouse's father.
- Kelly Hu as Jilly, Space Mouse's mother.
- Novie Edwards as Katie, Space Mouse's owner.
- Jason Lee as Splinter, a tiger who is Space Mouse's arch-nemesis and antagonist of the film.
- Kate McKinnon as Nellie, a cockatoo one of Space Mouse's friends.
- Hynden Walch as Misty, a European hedgehog one of Space Mouse's friends.
- Jacqueline Pillon as Brainy, a European rabbit one of Space Mouse's friends.
- Craig T. Nelson as Seamus, a German Shepherd Dog who is Space Mouse's best friend.
- Owen Wilson as Joey, a North American raccoon one of Space Mouse's friends.
- Mae Whitman as Joy, a house mouse who is Space Mouse's love interest.
- Sia as Ivy, a White-Tailed Deer who is Space Mouse's second best friend.
- Samuel L. Jackson as Sultan, an African spurred tortoise one of Space Mouse's friends.
- Andrew Kishino as Leonard, an African wild dog who the secondary antagonist of the film.
- Tom Conway as The Narrator.
- Don't Go Breaking My Heart – Elton John
- You're My Best Friend – Queen
- Happy Together – The Turtles
- Hotel California – Eagles
- Bad Day – Daniel Powter
- What a Wonderful World – Louis Armstrong
- Beyond the Sea – Robbie Williams
- Love Song by Sara Bareilles is played in the trailer.
- Beyond the Sea by Robbie Williams is played in the ending credits of the movie.
- Space Mouse first aired on Cartoon Network on October 13, 2010.
- Space Mouse first aired on Nickelodeon on May 14, 2013, on September 17, 2014 and on June 14, 2019.
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