After a daring escape from prison, Doctor Octopus forms an uneasy alliance with five other super villains ( Electro, Kraven the Hunter, Mysterio, Sandman and the Vulture) in the hopes of completely crushing their arch-enemy Spider-Man. In order to lure the unsuspecting superhero into their deadly game, the newly formed Sinister Six kidnap Betty Brant away from the "Daily Bugle" building, accidentally snatching Peter Parker's Aunt May in the process.
Issuing a challenge to the web-head, Spidey is told he must fight each of the villains one-at-a-time. If he can get past each villain in turn, he will receive a card telling him where to battle his next foe...each card he receives taking him one step closer to freeing Betty and May.
His first fight is against Electro at building #4 of Tony Stark's electrical plant. After using Electro's own power against him, Spider-Man moves on to do battle with Kraven and two of his trained leopards.
The web-slinger receives a shock next when he discovers his third fight will be contested against the X-Men! Come to find out, his opponents are actually robotic constructs set up by Mysterio. After tangling with that member of the Sinister Six, it's off for a dangerous bout against the Sandman in the confines of an air-tight metal box.
The only villain standing between Spidey and his ultimate confrontation with Doc Ock now is the Vulture, whom the wall-crawler must fight without the aid of his trusty web-shooters. Finally learning the location of the castle where Octopus is residing, Spider-Man arrives for the final battle. It's a decidedly nasty sparring match that starts with Spidey fending off a solo attack from Ock's mechanical arms...and winds up in a giant water-filled fishbowl against the reunited arms and their menacing master.
Victorious at last, Spider-Man frees Betty Brant and his Aunt before alerting the police to the whereabouts of the Sinister Six members (all of whom wind up in the same jail cell together to bemoan their defeat).
Along the way, there are plenty of special guest stars from the likes of the Fantastic Four and Dr. Strange (among others) as well as a gallery of Spider-Man's most famous foes. Next is an article on the "Secrets of Spider-Man". There are also small segments on Peter Parker's classmates and his residence, then the first-ever Amazing Spider-Man Annual ends with a humorous look at "How Stan Lee and Steve Ditko Create Spider-Man".