In an unexpected twist, the cherished exhibits at the American Museum of Natural History send the newest guard into a panic, much to the chagrin of Larry Daley, who is merely passing through. Out of the shadows emerge the ghostly figures of Theodore Roosevelt and Sacagawea, suggesting that Larry's son, Nick, would be the perfect choice to take over the guard position. After some internal conflict, Larry gives his reluctant approval.
Meanwhile, Nick is wrestling with his own aspirations, hoping to play the role of DJ at his school’s jazz recital to impress his crush, Mia. Though his teacher, Ms. Montefusco, recognizes his potential, Nick is convinced that he's fallen short, feeling like a musical failure. Larry attempts to bolster Nick’s spirits by explaining that while Nick will be covering for him at home, he’ll also be taking on the responsibilities of guarding the museum in Japan this summer. Though initially doubtful, Nick ultimately agrees after strong encouragement from his father and ex-wife, Erica.
As Nick reconnects with his eclectic gang—comprised of Rexy, Attila, Jedediah, Octavius, Laaa, Dexter the monkey, and Joan of Arc—he finds himself in charge of securing the basement storage room. However, the unsettling atmosphere and ominous relics prompt Nick to barricade the door with a statue and make a swift exit.
Meanwhile, the villainous Kahmunrah, having escaped captivity, seizes the magical tablet that animates the exhibits and plots to dominate the world. A heart-pounding chase breaks out across the museum, but Kahmunrah proves slippery, ultimately escaping with the tablet. Nick and his allies pursue him while Rexy takes on the task of safeguarding the museum.
Once outside, Joan experiences a vision indicating that Kahmunrah is headed to the Museum of Natural Art, where a special exhibition on the Temple of Dendur is taking place. The gang embarks on a chaotic quest through the art museum, where Kahmunrah, now backed by Seth, the God of Chaos, employs his powers to keep them at bay.
They discover a painting that acts as a portal to ancient Egypt and plunge into it. With dawn approaching, they traverse the Nile River, during which Nick begins to despair over his inability to capture Kahmunrah. However, his friends rally around him with encouragement, helping him regain his resolve. Their journey culminates at the Temple of Dendur, where they navigate dangerous traps before facing Kahmunrah and Seth, who intend to utilize the tablet’s power by playing three specific notes to unleash an army of jackals.
As the serene ambiance of the ancient temple erupts into chaos, Nick confronts his deep-rooted insecurities. In a bold and unconventional move, he reverses the sequence of the notes, harnessing the tablet's energy to disperse the vicious forces of Seth and Kahmunrah, who had been terrorizing the temple with their jackal minions. Victorious as the supernatural threats are vanquished, Nick and his friends make a daring escape just as the sun rises, bathing the scene in its warm glow.
As they dash away from the temple, Nick’s thoughts are drawn to a mysterious poster of the museum that Sacagawea had given him earlier. Realizing its significance, he leads his companions in a hasty retreat back to their world, barely escaping the consequences of the sun’s ascension.
With his self-assurance renewed and doubts relegated to the past, Nick seizes the chance to audition for the jazz recital once again—this time achieving spectacular success. As he basks in the limelight of his accomplishments, he finds a blossoming romance with Mia and embraces his newfound role as the museum’s night guard, a position that fulfills him and gives him a profound sense of belonging.
