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Barry B. Benson
- Voiced by: Jerry Seinfeld, Jaime Camil (Latin American Spanish)
- Alliterative Name: Barry B. Benson.
- Deadpan Snarker: Naturally, being voiced by Jerry Seinfeld.
- Didn't Think This Through: Did not think taking back the honey would cause so much damage to the environment.
- Did You Just Romance Cthulhu?: At the start of the movie Humans Are Cthulhu to bee's, yet he's attracted to Vanessa.
- Half-Dressed Cartoon Animal: He wears a yellow sweater with black stripes (though it blends in with his body so well that it's quite difficult to see it) as well as black and yellow sneakers.
- Heterosexual Life-Partners: With Adam.
- Insect Gender-Bender: He's a male worker bee with a stinger.
- 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.
Vanessa Bloome
- Voiced by: Renée Zellweger, Ana de la Reguera (Latin American Spanish)
- Deuteragonist: Of the movie.
- Friend to All Living Things: She owns her own flower shop and saved Barry's life when she had no obligation to do so.
- Giant Woman: Played with, in comparison to Barry and the other bee’s she is a giant.
- Meaningful Name: Her last name indicates her being a florist.
- Ms. Fanservice: She has a very attractive appeal, especially in Barry’s Imagine Spot.
- Nice Girl: The only human to help Barry. There's also how she saved his life.
- Platonic Life-Partners: With Barry.
Adam Flayman
- Voiced by: Matthew Broderick, René García (Latin American Spanish)
- Heterosexual Life-Partners: With Barry.
- Hidden Depths: He is really good at making paper boats.
- Rage Breaking Point: He gets so mad at Montgomery throwing insults at bees, he stings him in the butt, which ends up hurting Adam more than Montgomery. For the rest of the movie, his stinger is replaced with a pink toy sword in his abdomen.
- Sensitive Guy and Manly Man: The Sensitive Guy to Barry's Manly Man.
- Tritagonist: Of the movie.
Ken
- Voiced by: Patrick Warburton, Mario Filio (Latin American Spanish)
- Crazy Jealous Guy: He gets so jealous of Barry hanging out with Vanessa that he tries to kill him.
- Fantastic Racism: Because of his bee allergy, he hates bees, which veers into racism once it’s revealed bees are sapient.
- Jerkass: He tries to kill Barry even after learning that he’s intelligent.
- Large Ham: Comes with being voiced by Patrick Warburton. Ken screams a lot.
- Murder the Hypotenuse: Once it becomes clear that Barry is stealing his girlfriend, he tries to kill him with a rolled-up magazine.
- Only Sane Man: Parodied. He acts like he has a normal life and living in a world where bees are sapient and one steals his girlfriend drives him up the wall.
Layton T. Montgomery
- Voiced by: John Goodman (English), Bernard-Pierre Donnadieu (European French), Maynardo Zavala (Latin American Spanish)
- Ascended Extra: In the film, he only appears in the Honey Trial scenes, but he's the main villain of the tie-in video game.
- The Antagonist: He's Barry's main opponent in his goal to give honey back to the bees, also being a bee hater himself. However, Barry's as much of an antagonist of his own as he ends up disrupting the forces of nature and causing the plants to wither.
- Fat Bastard: An overweight lawyer who hates bees.
- 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.
- Large Ham: Just look how he reacts when Adam stings him.
- Minor Injury Overreaction: He responds this way when Adam stings him, rolling all over the floor and spilling paperwork everywhere.
Mooseblood Mosquito
- Voiced by: Chris Rock, Alfonso Obregón Inclán (Latin American Spanish)
- 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.
- Evil Lawyer Joke: He becomes a lawyer after the events of the movie, stating that he only needed a briefcase since he was "already a blood-sucking parasite."
- Gag Nose: His proboscis is placed on his face as if it were his nose.
- Goggles Do Nothing: He never uses the goggles sitting on his head.
- Insect Gender-Bender: Mooseblood is very clearly a male mosquito and apparently drinks blood, even though only female mosquitoes drink blood.
- Playing Possum: He and several other insects pretend to be dead while riding the windshield of a truck to avoid getting swiped away by the windshield wipers.