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With their mission to capture all 625 experiments and repurpose them on Earth completed, Lilo, Stitch, Jumba and Pleakley are honored as heroes by the Galactic Alliance. Jumba is given the confiscated key to his laboratory again, Pleakley is offered a post as chairman of Earth Studies at G.A.C.C. (Galactic Alliance Community College) and Stitch is made a Captain of the Galactic Armada and commander of his new commissioned ship BRB-9000 (BRB stands for "Big Red Battleship"). Lilo is made Galactic Federation Ambassador to Earth and sole guardian of Stitch's "cousins". Before they leave, Lilo gives Jumba her favorite Elvis record, Pleakley a paperweight (which is actually an Earth rock) and Stitch a necklace with a Ku Tiki (the Hawaiian god of strength). Meanwhile at prison, Gantu has decided that since he failed in capturing all of the experiments (except for 625), he will break Dr. Hämsterviel out of prison. He takes the two-man Space Shuttle, leaving 625 alone.
At Galactic Alliance HQ on Planet Turo, Stitch is assigned to recapture Hämsterviel. The ship he is assigned to is the BRB-9000 (Big Red Battleship to Stitch). However, he realizes that he misses Lilo. Pleakley is first overjoyed at his new position at G.A.C.C., until his assistant tells him that he is merely a supervisory professor—he will teach no classes. Jumba is also happy at first returning to many of his "Evil Genius" ways, but discovers that without anyone to show his accomplishments to, he is as unhappy as the others. Jumba's sadness is short-lived when Hämsterviel and Gantu burst in. Hämsterviel forces Jumba to create a new and more evil experiment to defeat the Galactic Alliance. The new creation has all of Stitch's powers and some others. Wishing to imitate Lilo in giving the experiments names, Hämsterviel names the creature Leroy.
Stitch arrives, seeking to capture Hämsterviel. After a fight that destroys Jumba's lab, Stitch is defeated when Pleakley appears at an unfortunate moment, distracting Stitch long enough for Leroy to lock him in a container. Hämsterviel reveals his master plan: to clone an army of Leroys to take over the Galactic Alliance. Before leaving for Turo, Hämsterviel locks Jumba, Pleakley and Stitch in Pleakley's space vehicle, and sends the ship into a black hole.
Back on Earth, Lilo makes the rounds to ensure that all of the "cousins" are still happy in their "one true place". After a confrontation with Mertle, Lilo decides to contact Stitch. Lilo realizes the only inter-galactic videophone on the planet available (she attempts to call Cobra Bubbles and fails) is in Gantu's ship. There, she finds 625, who is grouchy about being left behind by Gantu and asks to use the videophone. However, 625 says it is broken. It occurs to Lilo that 625 doesn't have a name and, after several sandwich puns, settles on "Reuben". Reuben then consents to help Lilo. Once the videophone is fixed, Lilo contacts the BRB-9000. The original Leroy makes a convincing impersonation of Stitch, using shape-shifting to disguise himself, but Lilo knows that Leroy is not Stitch because he does not have the tiki necklace. Hämsterviel decides on a new task for Leroy: to go to Earth and capture all of the other experiments so he may destroy them.
Lilo, sensing Stitch is in trouble, asks Reuben for help in fixing Gantu's Ship. After some reluctance, he agrees. As they leave, a Leroy clone crashes on Earth in Mertle's back yard.
As Jumba, Pleakley and Stitch head towards the black hole, Stitch escapes and frees the others. However, the navigational computer is locked on course for the black hole. Jumba says that the black hole will teleport them to a volcanic planet where they will be instantly vaporized, but if they can disrupt the event horizon, it will send them somewhere else. Pleakley pulls out the rock that Lilo gave him and asks if this will work. After Jumba says it is too heavy, Stitch takes a bite out of it, then crawls out the front of the vehicle and throws it at the black hole. There is a flash and the vehicle is sucked in.
On Earth, Leroy obtains Lilo's scrapbook of "cousins" and quickly captures all of them (along with Mertle).
Lilo and Reuben arrive at Turo, but they are too late. Hämsterviel has taken over using Leroy and orders Gantu to lock them up, then turn in his I.D. card and cape. Gantu captures Lilo and Reuben, but decides to release them because Hämsterviel fired him. After a close call with several Leroy clones, they are trapped. All looks lost until the G.A.C.C. van suddenly appears. With no time to explain, Lilo, Reuben and Gantu all climb in and head for Earth (Gantu manages to squeeze into the trunk compartment).
On Earth, the original Leroy has herded all the experiments into a large stadium, where "Alohapalooza," is scheduled to take place. There is a large group scene of the entire group of experiments. The BRB-9000 appears and Hämsterviel prepares to obliterate all of the experiments, until a timely appearance by Lilo, Stitch and the others destroys the BRB's primary cannon. Hämsterviel reveals that he brought along his Leroy army as backup. Stitch rallies his "cousins" and the experiments begin to battle. Despite some initial victories by the Experiments, it soon becomes apparent that the Leroys are more than a match for the good guys. Jumba remembers that he programmed a secret shut-down command into Leroy. When the original Leroy was created, Jumba was playing a recording of Elvis Presley singing "Aloha `Oe". If Leroy hears that song again, it will cause him to deactivate. Lilo, seeing the equipment set up for the concert, devises a plan. With Jumba controlling the sound, Pleakley on lights, and Gantu providing fireworks, Stitch appears on-stage in full Elvis attire. He begins to play "Aloha `Oe" with Lilo and Reuben singing along and some of the other experiments on instruments and, sure enough, the Leroys have violent seizures and shut down. His plan foiled, Hämsterviel is recaptured.
Back at Galactic Alliance HQ, the Grand Councilwoman calls Lilo and the others "heroes of the Alliance." Stitch, Jumba and Pleakley ask to be allowed to return to Earth with Lilo. The Grand Councilwoman accepts this, then asks Captain Gantu if he would like to be reinstated as a Captain of the Armada. Gantu agrees on the condition that Reuben be assigned as his galley officer.
Back on Earth, Lilo sets up for one last picture. Mertle arrives with Gigi (during the battle, Mertle learned that Gigi could talk and was one of Jumba's experiments). Though Mertle still thinks Lilo is weird, she says that Gigi wants to be part of the ʻohana. Lilo's last picture in the cousins album is of all of the experiments still on Earth, plus herself, Mertle, Jumba, Pleakley, Nani and Nani's boyfriend, David.
Meanwhile, Lilo has already found out where Leroy and his clones belong: in jail. Before the ending credits, the clones of Leroy happily dance to the Elvis Presley song "Jailhouse Rock", with an angry Dr. Hämsterviel tapping his foot. As the credits roll, a full list of Jumba's experiments 001 through 626, and the names they were given by Lilo, scrolls along the left side of the screen.
- Chris Sanders as Stitch, Leroy
- Daveigh Chase as Lilo Pelekai
- Tia Carrere as Nani Pelekai
- David Ogden Stiers as Dr. Jumba Jookiba
- Kevin McDonald as Agent Pleakley
- Kevin Michael Richardson as Captain Gantu
- Jeff Bennett as Dr. Hämsterviel, Fibber, Slick, Ace, Clyde
- Rob Paulsen as Reuben, Squeak, Remmy
- Zoe Caldwell as Grand Councilwoman
- Ving Rhames as Cobra Bubbles
- Liliana Mumy as Mertle Edmonds
- Tara Strong as Angel, Belle, Houdini, Pleakley's assistant
- Frank Welker as Sparky, Sprout, Mr. Stenchy, Kixx, Holio
- Tress MacNeille as Bonnie, Poxy, Gigi, Richter, Topper, Felix, Melty, Amnesio, Cannonball, G.A.C.C. van computer
- Nancy Cartwright as Shortstuff, Phantasmo
- Bobcat Goldthwait as Nosy
- Jim Cummings as Heckler
- Lili Ishida as Yuki
- Jillian Henry as Elena
- Kali Whitehurst as Teresa
- Jason Scott Lee as David Kawena
- In one shot of the crowd of experiments at the stadium, you can briefly see The Lion King's Timon and Pumbaa standing in the crowd.
- The coordinates for the black hole are "[tel:12-21-9-7-9]", which is a reference to the release date of the film The Black Hole (December 21, 1979).
- Shrink, Kernel, Gotchu, Poki, Forehead, Frenchfry, Zawp, Mary, Skip, Stamen, Mulch, Squeak, Fudgy, Snafu, Bugby, Nosox, Retro, Poxy, Shush, Belle, Sproing, Ace, Wishy-Washy, Tickle-Tummy, Lax, Morpholomew, Heckler, Nodessertro, Manners, Ploot, Flame, Huggo, Wrapper, Digger, Blowhard, Derrick, Woops, Sinker, Zap, Cooler, Chortle, Ruinit, Vialet, Crystallene, Reuben and 627 do not appear in the group photo.
- There are many duplicates or recolors and slight edits of existing experiments seen during the battle, along with some that aren't experiments at all. You can see multiple orange replicas of Angel, duplicates of Nosox, Dupe, Crystallene, Yaarp, Hunkahunka, Digger, many different colored replicas of Cannonball, Snooty, Kernel, Nosy, Slick, a blue Deforestator and many others. It is possible that it would be too complicated to design them individually, so they did that.
- When Leroy is capturing experiments, Drowsy and Backhoe are both the same shape when they're blasted into nets. Also, their feet disappear instead of leaving the ground as the net traps them into it.
- Cannonball and Gigi have powers that were never used in the series: Cannonball having the ability to roll into a ball in midair, and Gigi having the ability to talk.
- Cannonball doesn't hold onto his feet when bouncing on his butt.
- Tickle-Tummy was somehow forgotten to be added on the list of experiments at the end credits, and most fans thought Tickle-Tummy was 628, but Tickle-Tummy is actually 275.
- Fudgy's number was originally "119", but somehow it changed to "054" on the list of experiments.
- Stopgo's number was originally "239", but somehow it changed to "102" on the list of experiments.
- Timon and Pumbaa appear in the movie even though they are not even in the Lilo and Stitch series.