- Dr. John Dolittle as Gru
- Grandpa Archer Dolittle as Dave the Minion
- Dr. Mark Weller as Tim the Minion
- Calloway as Dr. Nefario
- Lucky the Dog as Paul the Minion
- Rodney the Guinea Pig as Bob the Minion
- Lisa Dolittle as Margo
- Maya Dolittle as Agnes
- Charisse Dolittle as Edith
- Possum as Mr. Perkins
- Female Pigeon as Miss Hattie
- Skunk as Vector
- and more
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