* Steve Carell as Gru, the world's former #1 supervillain and the protagonist of the film. He at first only adopted the girls so he could steal the shrink ray from Vector, but later warms up to the girls and finally becomes their adoptive father.
* Jason Segel as Vector (a.k.a. Victor Perkins), the world's new #1 supervillain and the primary antagonist of the film. He has a penchant for inventing weaponry that launches underwater life
* Russell Brand as Dr. Nefario, Gru's elderly, hearing impaired scientist assistant. It is hinted that he is Gru's father, but Gru only refers to him as "good friend."
* Elsie Fisher as Agnes, the youngest girl of the group of orphans. She is known for being addicted to unicorns and likes Gru from the start
* Dana Gaier as Edith, the middle girl of the group of orphans. She likes gross things and wears a pink beanie. She is the only one of the girls to think that Vector's and Gru's inventions and weapons were "cool"
* Miranda Cosgrove as Margo, the oldest girl of the group of orphans. She is, for a majority of the film, untrusting of Gru. But she later learns to grow close to Gru and her sisters feel the same way
* Julie Andrews as Gru's mother. In several of Gru's flashbacks, he showed her his plans to go to the moon, but she ignores everything he said
* Will Arnett as Mr. Perkins, the president of the Bank of Evil, Vector's father, and the film's secondary antagonist.
* Kristen Wiig as Miss Hattie, mistress of a local orphanage, Miss Hattie's House of Girls. For the most part she seems to hate her job and sees the girls more as an irritation than anything else
* Pierre Coffin as Tim / Bob / Mark / Phil / Stuart, five of Gru's minions
* Chris Renaud as Dave, one of Gru's minions who fired a missile launcher during the minion gathering
* Ken Jeong as Talk Show Host
* Danny R. McBride as Fred McDade, Gru's next door neighbor
* Jack McBrayer as Tourist Father / Carnival Barker
* Mindy Kaling as Tourist Mother
* Jemaine Clement as Jerry, one of Gru's minions
* Rob Huebel as Anchorman / Newscaster
* Ken Daurio as Egyptian Guard
* Philip Mathew as Second Egyptian Guard and himself
* Additional Voices as all of Gru's minions
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