After Ben Reilly is killed by Norman Osborn's Goblin glider, Spider-Man battles revenge against his old enemy. However, in the midst of the battle, Spider-Man accidentally webs one of the Goblin's pumpkin bombs to his hand. The Goblin shouts in anger that they've doomed them both, but Spider-Man just claims that he's only doing his job, for his family, and for the whole world just as the bomb explodes and they are consumed in light.
15 years later, Peter Parker, now a one-legged forensic scientist working for the NYPD, awakens in the middle of the night from a nightmare reminiscing the battle. He then goes in and checks up on his daughter, May, who is sound asleep. The next day, Peter drives his daughter to school and drops her off. He then wishes her a good day at school and drives to work. During class, however, May begins to develop a sixth sense and grows nauseous. Later that evening, during a basketball game, May performs a triple somersault and jumps 13 feet into the air, throwing the ball and shattering the backboard. Peter just looks on in shock, believing that his greatest fears have come true. Afterwards, May and Peter head home. There, Peter reveals to his daughter about his secret past as Spider-Man, and the real reason he lost his leg. Afterwards, May begins pondering her possible powers and decides to test them, successfully managing to scale her bedroom wall. May then begins to design a costume for herself, based off of Ben Reilly's costume, and experiment with her powers, eventually uncovering her power to spin webs.
Meanwhile, at an abandoned Oscorp warehouse, a lone woman begins modifying and enhancing the Green Goblin's armor and weapons, all while musing Spider-Man's "inevitable" death by her hand.
The following day, at school, May decides to test her powers in the school gym after hours. She finds that her speed and agility have been enhanced beyond comprehension. Later that evening, she goes out on her first patrol as a costumed vigilante, taking on the name and persona of Spider-Girl. Finding a mugger in Harlem, May quickly makes short work of the criminal and leaves him for the police. The woman in distress thanks May for her actions and gets away. May then swings to a high location and ponders her victory. May then swings home, only to be confronted by her father. Peter orders May to never play hero again and confiscates her costume. Frustrated, May decides to go against her father's wishes and makes a makeshift costume instead, secretly going on patrol.
The next day, Peter is attacked by a woman who bears a striking resemblance to the Green Goblin. The woman orders Peter to find Spider-Man, as he was his photographer years ago, or she will go after everyone Peter cared for. Angered, Peter dons his Spider-Man costume and chases the woman to the Empire State Building, only to be defeated due to his dulled powers.
May hears of this on the news and, witnessing her father, decides to go against his wishes and reclaims her original costume and swings to the scene of the crime. Upon arriving, the woman announces to Spider-Girl as Fury, the Goblin Queen. Enraged by the sight of her father being held captive, Spider-Girl explodes into battle and angrily fights Fury to a standstill. However, Fury then gives an ultimatum to Spider-Girl; fight her or save her father. May chooses to save her father, but Fury uses the opportunity to kill her and Spider-Man. However, a police helicopter, siding with Spider-Girl, fires at Fury, forcing her to move away. May then gets her father to safety.
Enraged, Fury returns with a vengeance, grabs Spider-Girl, and throws her into an abandoned apartment building. The two then have a very one sided fist fight, with May almost getting beaten to death. However, as Fury muses how she will kill Spider-Man once she is done with his daughter, and enraged Spider-Girl overpowers and defeats Fury. Fury, however, calls on her glider and escapes.
Days later, Peter and May visit the cemetery, where they visit Mary Jane Watson's grave, and Peter looks to his Uncle Ben's gravestone as well. After returning home, Peter announces to May that after her performance against Fury, he approves of her responsibility as Spider-Girl. To further compensate this, Peter gives May a brand new costume to replace the one she damaged during her fight with Fury. Filled with hope, May hugs her father and tries on her costume before swinging away to have more adventures.