In 1957, nineteen years after The Last Crusade, Indiana Jones and his partner George "Mac" McHale are kidnapped in Nevada by Soviet agents under Colonel Dr. Irina Spalko. The Soviets infiltrate warehouse labeled "Warehouse 51" and force Jones to locate an alien corpse with a crystal skull, recovered ten years earlier. Upon its discovery, Mac reveals he is a double agent working for the Soviets. Jones escapes, unsuccessfully attempts to retrieve the skull, and in a fight with Spalko's sadistic henchman, Colonel Antonin Dovchenko, they both fall onto a rocket sled, which ignites and speeds them away. Jones staggers away and, still pursued, arrives in a model town at the Nevada Test Site, minutes before an atomic bomb test, and takes shelter in a lead-lined refrigerator. Jones is rescued, decontaminated, and arrested by FBI agents, who suspect him of working for the Soviets; and though freed (also bailed out by Team Griffin/Eeveelution and their friends) on the recommendation of General Ross, who vouches for him, he is put on indefinite leave of absence from Marshall College, also at the cost of the dean having to resign to keep Indiana's job at the college.
Jones and our heroes are approached by greaser Mutt Williams (Shia LaBeouf), who tells him that Harold Oxley had found a crystal skull in Peru, suffered a mental breakdown, and was later kidnapped. In return, Jones tells Mutt about the legend of crystal skulls found in Akator. Mutt gives Jones a letter from his mother, who is also held captive, containing a riddle written by Oxley in an ancient Native American language. KGB agents and Team Galactic try to take the letter, but Jones, our heroes, and Mutt evade them and reach Peru. At the local psychiatric hospital, Oxley's scribbles on the walls and floor of his cell lead them to the grave of Francisco de Orellana, a Conquistador searching for Akator. They discover the skull at the grave, with Jones reasoning that Oxley had returned it there.
On leaving the grave site, Jones, our heroes, and Mutt are captured by Mac, the villains, and the Soviets and taken to their camp in the Amazon jungle, where they find Oxley and Mutt's mother, Marion Ravenwood, who later reveals that Mutt is Jones' son, Henry Jones III. Jones berates her for not convincing him to finish school. Mac tells Jones he is really a CIA double-agent to regain Jones' trust. Spalko believes that the crystal skull belongs to an alien life form and holds great psychic power, and that finding more skulls in Akator will grant the Soviets the advantage of psychic warfare. And Ernie tells our heroes he's looking for a rare lightsaber, not from a Jedi, but from somebody else. Spalko uses the skull on Jones to enable him to understand Oxley and identify a route to Akator. Jones and his four allies escape with the skull into the Amazon. They elude giant ants (possibly a relation of Eciton burchellii, which Jones refers to as "siafu"), and after Dovchenko loses a fight with Jones, he is devoured by the ants. Jones, our heroes, and his allies survive three waterfalls in a GAZ 46 amphibious vehicle (which gets destroyed after the third drop), as many of the Soviets fall from a cliff while trying to pursue them. But however, they've managed to capture Blythe, the pets, and Hanah! Jones and Oxley then identify a rock formation that leads them to Akator, unaware that Mac is still loyal to Spalko and has been dropping transceivers to allow the surviving Soviets to track them.
They escape the city's guardians, gain access to the temple, and find it filled with artifacts from many ancient civilizations. Indy believes the aliens - in fact, inter-dimensional beings - were "archaeologists" studying the different cultures of Earth, and Mac remarks that there's not a museum in the world that wouldn't sell its soul for the collection inside the temple. The five enter a chamber containing the crystal skeletons of thirteen enthroned skeletal crystal beings, one missing its skull. Spalko arrives and presents the skull to this skeleton. It suddenly flies from her hands to the skeleton and rejoins, whereupon the aliens reanimate and telepathically offer a reward in ancient Mayan through Oxley. A portal to their dimension becomes activated, and Spalko demands knowledge equal to the aliens'. The thirteen beings fuse into one, and in the process of receiving the overwhelming knowledge, Spalko is disintegrated and sucked into the portal. And then after the battle with the droids and Death Troopers, Bellwether and the other villains have been killed, except Ernie who still wields Darth Vader's lightsaber. And he then tries to fight Kitty, Hanah, and Lois but is no match for their moves of Makashi and Ataru, and even Hanah's moves. And then Blythe and Dawn gun point him to drop the lightsaber, but Ernie refuses and clashes with them, and then finally Brian cuts off his arms and grabs the lightsaber in his hands, and decapitates Ernie with it, which surprises the girls.
Indy, Marion, Mutt, and Oxley - now released from the skull - escape with our heroes, while the Soviets are also drawn into the portal. Mac is caught in the pull while trying to scrounge some of the treasure, and even though Indy offers him the whip to pull him to safety, he replies wink of his eye, "Jonesy, I'm gonna be all right," lets go and is pulled in. The survivors watch as the temple walls crumble, revealing a flying saucer rising from the debris, which vanishes into the "space between spaces" while the hollow in the valley floor left by its departure is flooded by the waters of the Amazon.
The following year, Indy is reinstated at Marshall College and made an associate dean. He and Marion are then married in a church. As the wedding party leaves the chapel, a gust of wind blows Indy's brown fedora off the coat rack and deposits it at Mutt's feet. Mutt picks it up and is about to don it before Indy takes it from him and puts it on with a grin.
- The Griffin Family (Peter Griffin, Lois Griffin, Meg Griffin, Chris Griffin, and Stewie Griffin), Vinny Griffin, Glenn Quagmire, Cleveland Brown, Joe Swanson, Little Bear, Duck, Cat, Owl, Hen, Emily, Lucy, Doug Funnie, Skeeter Valentine, Belle, Dudley Puppy, Kitty Katswell, Hanah Streaker, Roberta Bravo, Judy Hopps, Nick Wilde, Blythe Baxter, Zoe Trent, Pepper Clark, Penny Ling, Sunil Nevla, Vinnie Terrio, Minka Mark, Russell Ferguson, Gail Trent, and Mitzi guest star in this film.
- Ernie the Giant Chicken, Sideshow Bob, Bobby Briggs, James Woods, The Dazzlings, Bellwether, Gaston, and Team Galactic (Mars, Jupiter and Saturn) will work for Irina Spalko in this film.
- The storyline concludes in Brian and the Eeveelution Family's Adventures of Indiana Jones 5.