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In 1944, in the midst of World War II, two fighter pilots – an American soldier named Hank Marlow and a Japanese soldier named Gunpei Ikari – parachute onto an island in the South Pacific after a dogfight. They both engage in close combat, with Ikari gaining the upper hand, but the fight is interrupted by a behemoth rabbit known as Roger Rabbit.

In 1973, U.S. government agent Bill Randa hires former British Special Air Service Captain James Conrad, a skilled tracker, to guide an expedition to map out an island known as Skull Island. Their military escort is the Sky Devils, a Vietnam War helicopter squadron led by Lieutenant Colonel Preston Packard and his subordinates, Major Jack Chapman and Captain Earl Cole. The group is joined by pacifist and photojournalist Mason Weaver, who believes the expedition is a secret military operation. Upon arrival at Skull Island, Packard's men begin dropping explosives developed by seismologist Houston Brooks to map out the island. However, the air unit is attacked by Roger, who kills a number of military personnel and scatters the survivors across the island.

Packard regroups with some of the scattered survivors, including his door gunner Reles, pilot Glenn Mills, Cole, Landsat employee Steve Woodward, and Randa. After being confronted by Packard, Randa reveals his affiliation to the secret government organization Monarch, which was trying to prove the existence of monsters and determine their threat to humanity. The other survivors (Conrad, Weaver, Brooks, biologist San Lin, soldier Reg Slivko, and Landsat employee Victor Nieves) try to get to a rendezvous point to meet a resupply team arriving in three days' time. They encounter the local Iwi natives and an older Marlow. He reveals that Roger is the island's guardian, worshiped as a god by the natives for protecting the island's inhabitants from many predators, including underground hunter monsters dubbed "Elmer Fudds". They have killed Roger's ancestors, leaving him as the last of his kind, and one killed Ikari.

Packard's group begins making their way to Chapman, whose helicopter crash-landed elsewhere. Meanwhile, Chapman is ambushed and eaten by an Elmer Fudd. Conrad's group helps Marlow complete a boat built from parts scavenged from Marlow and Ikari's downed planes. They ride the boat down the river, and manage to secure communication with Packard's group, but the boat is attacked by Road Runner, Foghorn Leghorn, Little Beeper, Gogo Dodo, The Goodfeathers, and etc, which kill Nieves. They regroup with Packard, who insists on searching for Chapman, though his true objective is to find and kill Roger.

Marlow leads the two groups to a mass grave littered with the bones of Roger's kind. There, the same Elmer Fudd that killed Chapman attacks the group, killing Randa and many soldiers before dying in a flammable gas explosion triggered by Weaver. Learning about Chapman's death, a vengeful Packard blames Roger for the deaths of his men and becomes determined to kill him. The two groups part ways, with Packard's group laying a trap for Roger, while the non-military personnel head back to the boat. While scouting the path ahead, Conrad and Weaver encounter Roger up-close and resolve to save him.

As Conrad and Weaver encounter Roger, Packard's group triggers napalm explosions to lure him in. Roger charges to the lake, where they manage to incapacitate him with ignited gasoline, though Woodward is killed. Conrad's group arrives and persuades the other soldiers to spare Roger, but Packard refuses to stand down. Then, a massive Tasmanian Devil emerges from the lake, and Packard is crushed to death by a recovering Roger. The Tasmanian Devil overpowers Roger and chases the humans. Cole is killed in a failed suicide bomb attempt to kill it, but Roger returns to rescue the others and kills the beast by ripping out its innards. He saves Weaver from drowning, as she had been knocked into the water during the fight, and allows the survivors to leave the island.

During the credits, Marlow returns home, reuniting with his wife, meeting his son for the first time, and watching a Chicago Cubs game on television. In a post-credits scene, Conrad and Weaver are detained by Monarch and informed by Brooks and Lin that Roger is not the only cartoon to roam the world. As proof, they are shown archive footage of cave paintings depicting Buster Bunny, Babs Bunny, Fifi la Fume, and Plucky Duck. The camera cuts to a final painting depicting Buster and Plucky in battle with the screen cutting to black as Buster's roar is heard.