- Two references to The Beatles include the song "Here comes the Sun," and the Flight Controller saying "Coo-Coo-Ca-Choo," as the plane flies and settles in a bee-like fashion.
- In the first scene they show a collection of white sneakers that Barry owns. In real life, Jerry Seinfeld is said to have a collection of more than 500 sneakers and they are all white.
- According to Jerry Seinfeld, while having lunch at Steven Spielberg's house, he first mentioned the idea for the movie as a joke, but Spielberg loved the idea.
- Both of Barry's nemesis are voiced by stars of films produced by Dreamworks nemesis, Pixar. John Goodman, the voice of the opposing council, had the lead role in Monsters, Inc.. Patrick Warburton, the voice of the human who almost killed Barry, was the title character in Pixar's Buzz Lightyear of Star Command.
- The first two movie trailers had Jerry Seinfeld trying to make a live action Bee Movie. After watching the disastrous results, Steven Spielberg asks, "Why don't you just make it a cartoon?"
- Bees in the real world have a very different biology than the bees in this movie. In the real world, male bees don't contribute at all to the honey-making process, which is done entirely by females. Male bees are drones, and just hang around the hive to mate with the queen. Drones have no stingers - that's the female reproductive organ and self-defense method - though bees do tend to die after stinging, as the stinger (which has tiny barbs on it) rips free of the body as the bee flies away. Also, male mosquitoes do not drink blood, only female mosquitoes do, as a protein source for their eggs, not as sustenance for themselves. Furthermore, insects have 6 legs, not 4. Lastly, they do not have teeth, clothing or the ability to mimic human speech.
- In some areas, release prints were delivered to theaters with fake titles: "Flower" and "Jocks".
- The actual address of the fictitious Vanessa's Flower Shop is at the corner of 67th W and Columbus, New York, New York.