Five years after the retirement of Agent K and the defeat of Edgar the Bug from ''Men in Black'', the secret New York City-based agency that monitors and regulates extraterrestrial life residing on Earth, Agent J–K's former partner and hand-picked replacement–is called to investigate the murder of an alien, Ben, at his pizzeria. The waitress, Laura Vasquez, tells him that the murderers are Serleena, a shapeshifting, plant-like Kylothian who has taken the form of a lingerie model, and her two-headed servant Scrad. Laura says they were looking for something called the Light of Zartha. J is strongly attracted to Laura, and in violation of MiB rules, does not Neuralyzer her to erase her memories.

Following a ghost invasion of Manhattan, paranormal enthusiasts Erin Gilbert and Abby Yates, nuclear engineer Jillian Holtzmann, and subway worker Patty Tolan band together to stop the otherworldly threat.