- Voiced by: Renée Zellweger
- Friend to All Living Things: She owns her own flower shop and saved Barry's life when she had no obligation to do so.
- Meaningful Name: Her last name indicates her being a florist.
- Nice Lady: The only human to help Barry. There's also how she saved his life.
- Platonic Life-Partners: With Barry.
- The Hero: The main protagonist.
Barry B. Benson
- Voiced by: Jerry Seinfeld
- Alliterative Name: Barry B. Benson.
- Deuteragonist: Of the movie.
- Didn't Think This Through: Did not think taking back the honey would cause so much damage to the environment.
- Heterosexual Life-Partners: With Adam.
- Nice Guy: A laid-back guy who sets out to help his colony. And then to fix the damage he caused to the environment.
- Platonic Life-Partners: With Vanessa.
- Sensitive Guy and Manly Man: The Manly Man to Adam's Sensitive Guy.
- Voiced by: Matthew Broderick
- Disney Death: During the climax, he gets killed when he stings Montgomery. But he is doing alive and well when Barry visits him.
- Heterosexual Life-Partners: With Barry.
- Sensitive Guy and Manly Man: The Sensitive Guy to Barry's Manly Man.
- Tritagonist: Of the movie.
- Voiced by: Patrick Warburton
- Big Bad: He become this of the movie.
- Big Bad Duumvirate: He teams up with Layton T. Montgomery to kill Barry.
- Bitch in Sheep's Clothing: At first, he was a hero and Vanessa's boyfriend. But he secretly the true mastermind behind the Honey Trail.
- Dragon-in-Chief: He becomes this to Montgomery in the film.
Layton T. Montgomery
- Voiced by: John Goodman
- Advertised Extra: Despite being the Big Bad Wannabe, he gets a few brief scenes of screen time at the middle of the film. His actor's name appears in the opening credits of the film.
- Ascended Extra: He appears in the Honey Trial scenes but is the main villain of the tie-in video game.
- Big Bad Duumvirate: With Ken.
- Big Bad Wannabe: He becomes a bigger threat in the movie, but he is still Ken's top henchman.
- The Dragon: To Ken.
- Jerkass Has a Point: While Mr. Montgomery's court tactics aren't very morally ethical, his assertions are proven true when nature begins to suffer as a result of the bees winning the honey trial and subsequently quitting their jobs.
- Voiced by: Chris Rock
- Advertised Extra: Despite appearing in the promo photos and trailers, he has five minutes of screen time, making him a minor character. He even has his own wallpaper and his actor gets billed last in the opening credits.