Peter Parker e seu professor de ciência da faculdade Dr. Curt Conners explorar os métodos neogênicos de regeneração de membros. Mas ao experimentar em si mesmo, Connors acidentalmente torna-se o Lagarto, um meio-homem, meio-réptil, que aterroriza Manhattan e, posteriormente, sequestra sua própria esposa. A tarefa do Homem-Aranha é duplamente difícil porque ele não quer machucar o bom amigo que ele sabe é preso dentro do monstro... mas ele deve parar a besta desumana que está fora de controle.

Norman Osborn hires Spencer Smythe to design a machine capable of destroying Spider-Man - the Black Widow Spider Slayer. But when Flash Thompson puts on a Spider-Man costume as a party gag, the deadly machine spots him, and he's carried off in Peter's place.

With Spencer Smythe dead, his son Alistair turns to the Kingpin to help seek his revenge on Spider-Man and others he deems responsible for his father's death. He captures, Eddie Brock, J. Jonah Jameson, Norman Osborn, and Flash Thompson. He then turns his sights on Spidey and sends three slayers after him. Spidey only manages to hold his ground while fighting them individually. However, he is not so lucky when they combine into a super slayer and carry him off to Smythe.

The Master of Illusion, Mysterio has fooled the city and J. Jonah Jameson into thinking Spidey has turned to crime. However Spidey manages to find Mysterio only to be brought to a trap of special effects.

J. Jonah Jameson hires Dr. Farley Stillwell who is a raining genius in genetic engineering, to make a new super hero that can match and defeat Spider-Man. With a willing participant, Mac Gargain, they engineer the DNA of a scorpion and him together to form a new hero, The Scorpion. However, when Mac thinks they have turned him into a freak, hero turns to monster and he goes on a rampage. Spider-Man is then forced to step in to stop creation from killing creator.

Robbie has set Peter up with a job interview with a researcher by the name of Dr. Mariah Crawford. However, when he gets to her lab, he finds her under attack by a monstrous man who deems himself Kraven the Hunter. Spider-Man leads Crawford away from Kraven to the roofs. There, Crawford tells Spider-Man that Kraven was not always that way, that in fact she loved him as Sergei Kravenoff. Crawford tells of an accident years back that would have killed Sergei had she not used a dangerous potion that saved his life and gave him the strength and instinct of an animal, but turned him mad. Meanwhile, while looking for Crawford at her apartment, Kraven learns of her connection to Robbie and kidnaps him. He then threatens to kill Robbie if Spider-Man does not come to him with Crawford.

A space shuttle piloted by J.J.'s son, John Jameson, crash lands on the Tri-Borough Bridge carrying two strange meteors -- One that could provide a new nuclear fuel and one that contains an alien substance. The first is stolen by the Kingpin's henchman, the Rhino, just as Spider-Man arrives to save Jameson and the co-pilots. When the Rhino sends the aircraft off the bridge, Spider-Man emerges from the water with a dark stain covering his costume. Exhausted, he heads for home, only to awaken hanging from a skyscraper, now clothed in a black version of his costume.

Spider-Man goes after Eddie Brock for labeling him as he is now being stalked by bounty hunters at every turn. After being reassured by Mary Jane that he has changed, he seeks help from Dr. Curt Connors who discovers that the suit is alive!! And Peter is the host organism!

The Kingpin is furious at Spidey and sends the Shocker and later the Rhino out to finish him off with the aid of another new invention of Alistair Smythe. Meanwhile, Eddie Brock, as the symbiote creature Venom, is busy playing cat and mouse with Spider-Man, intruding on Peter's private life and making it clear he knows the web-slinger's secret identity. Peter soon realizes that the pumped-up Eddie as Venom is stronger than he is. Worse than that: Spidey's spider-sense doesn't work around him!

Norman Osborn hires hitman and thief, the Hobgoblin, to take out Osborn's "silent" partner, Wilson Fisk (the Kingpin). When he fails in the attempt, the Hobgoblin changes sides, kidnapping Norman's son, Harry. Harry just happens to be best friends with Peter Parker.

When Aunt May is slightly injured by one of the Hobgoblin's pumpkin bombs during the kidnapping of Harry, Spider-Man enters the fray with a vengeance. After several battles, the trail leads Peter to Oscorp and Harry's own father. For Peter, rescuing his friend is easy compared to explaining to Harry that his own father is involved.

An international spy and hitman known as the Chameleon plans to assassinate two major diplomats on the brink of signing the peace proposal of the century. S.H.I.E.L.D. director Nick Fury solicits J. Jonah Jameson's assistance in an effort to prevent the "human shape-shifter" from derailing the proposal signing. But only Spider-Man with his super keen "spider-sense" can discern who is really whom and stop the criminal before it's too late.