The film follows Joe Shimamura, a race car driver kidnapped by Black Ghost and modified into a cyborg dubbed "Cyborg 009". He manages to escape along his fellow 00 Cyborgs and former Black Ghost scientist Dr. Gilmore. Together, they work to take down Black Ghost and stop their nefarious plans.
It was followed up in 1967 with a sequel titled Cyborg 009: Monster Wars.
- Age Lift: 007 was changed into a small child for this movie, with the reasoning being so that young children would have someone to identify with.
- Alien Blood: When 009 is being operated on, his blood is shown to be dark green.
- Bad Boss: A Black Ghost official kills two of his subordinates for failing to clean up evidence at the scene of Joe's kidnapping. Ironically, said official later gets Bad Boss'd by the leader of Black Ghost after he fails to stop the 00 Cyborgs from escaping. And the aforementioned "leader of Black Ghost" ends up getting killed by his boss when he gets cornered by the 00 Cyborgs.
- Brainwashed and Crazy: 003 gets captured and brainwashed by Black Ghost at one point. She gets better.
- Demoted to Extra: Aside from 009, the other 00 Cyborgs act more as secondary characters and don't even get their backstories explained.
- Skull is absent from the film, with the leader of Black Ghost being a dark green figure resembling a demon.
- The 00 Cyborgs also encounter a machine bearing a heavy resemblance to 0011, but it is never named.
- The true leader of Black Ghost is one to the three brains inside the Demon Statue that are the true leaders of Black Ghost in the manga, though its design bears more resemblance to 0012.
- Law of Chromatic Superiority: Instead of red, all of the 00 Cyborgs' uniforms are colored gray, except for 009's, which is white, and 003's which is pink. Naturally, 009 is the main character while 003 is his Love Interest.
- The Man Behind the Man: The character we initially think is the leader of Black Ghost turns out to be subordinate to a Brain in a Jar.
- Race Lift: 009 being biracial is never brought up in the film.
- Shout Out: The leader of Black Ghost's Right-Hand Cat bears a resemblance to Tom. For bonus points, 007 at one points transforms into a mouse that resembles Jerry.
- Wham Line: "But, I am not the boss of Black Ghost!"