In 2379, two hundred years after the events of Alien 3, military scientists on the space vessel USM Auriga create a clone of Ellen Ripley using DNA from blood samples taken before her death. The Alien queen's DNA was mixed in with Ripley's, and the clone grows up with an embryo inside it. The scientists extract the embryo, raise it and collect its eggs. The Ripley clone is kept alive for further study. As a result of the alien's DNA inside her, she develops enhanced strength and reflexes, has acidic blood and a psychic link with the Aliens. Also, the alien's genetic memory allows the clone to have some of Ripley's memories.
A group of mercenaries, Elgyn, Johner, Christie, Vriess, Hillard and Call, arrive at Auriga on their ship Betty. They deliver several kidnapped humans in stasis. The military scientists use the humans as hosts for the Aliens, raising several adult Aliens for study.
The Betty crew soon encounters Ripley. Call recognizes her name and tries to kill her, suspecting she may be used to create more Aliens. The Aliens have already matured and escape confinement by killing off one of their own and using the acidic blood to burn through their enclosures. They damage the Auriga and kill some of those people who do not evacuate, including General Perez and Elgyn. Another crew member is coccooned. Military scientist Dr. Wren reveals that the ship's default command in an emergency is to return to Earth. Realizing this will unleash the Aliens on Earth, Ripley, the mercenaries, Wren, a Marine named DiStephano and surviving Alien host Purvis decide to head for the Betty and use it to destroy the Auriga. Along the way, Ripley encounters the grotesque products of failed attempts to clone Ripley. The surviving one begs Ripley to mercy kill her, and she complies.
As the group makes their way through the damaged ship, they swim through a flooded kitchen. They are chased by two Aliens. One is killed, while the other snatches a weak Hillard. As they escape the kitchen, the Alien returns and blinds Christie, who sacrifices himself to kill the Alien so the others can escape. After Wren betrays the group, Call is revealed to be an android. Using her ability to interface with the Auriga's systems, Call sets it on a collision course with Earth, hoping to destroy the Aliens in the crash. She cuts off Wren's escape route, and directs the Aliens towards him. Ripley is captured by an Alien, while the others head for the Betty. Wren shoots Purvis and takes Call hostage, demanding that she abort the collision. An injured Purvis attacks Wren and forces his head to his chest just as the Alien embryo he is carrying bursts through his ribcage, causing it to go through Wren's head too, killing them both. The survivors shoot and kill the young alien.
Ripley is taken to the Alien nest, where the Queen, now possessing a womb as a result of the genetic mixture, gives birth to a Newborn, a Xenomorph with human traits. The hybrid Alien recognizes Ripley as its mother, kills the queen Alien and Dr. Jonathan Gediman, a scientist previously captured and cocooned. Ripley takes advantage of the distraction to escape and makes her way to the Betty.
The Newborn reaches the Betty and attacks Call. It kills DiStephano when he tries to help her. Ripley finds her way onto the ship and saves Call by distracting the Newborn. Using her acidic blood, Ripley melts the glass of a window and pushes the Newborn towards the hole. The Newborn is then violently sucked through the hole due to decompression as Ripley tearfully watches on.
The countdown on the Auriga continues as the survivors escape in the Betty. The Auriga collides with Earth, causing a large explosion. Call and Ripley look down at Earth, and when Call asks what Ripley wants to do next, she says, "I'm a stranger here myself." In an alternate ending that is used in some extended adaptations, the Betty lands in a ruined Paris.
- Knockout, Dolphy, Puffer, Rachel, Mighty Joe, Daylight, Jenny, Pete, Kevin, July, Skarloey, Rheneas, Sir Handel, Peter Sam, Rusty, Duncan, Duck, Donald and Douglas, Oliver, Toad, Stepney, Daisy, Team Griffin/Eeveelution, The Griffin Family, Vinny Griffin, Glenn Quagmire, Cleveland Brown, Joe Swanson, Little Bear, Duck, Cat, Owl, Hen, Emily, Lucy, Doug Funnie, Skeeter Valentine, Natsu Dragneel, Lucy Heartfilia, Gray Fullbuster, Erza Scarlet, Happy, Carla, Dudley Puppy, Kitty Katswell, SpongeBob SquarePants, Patrick Star, Squidward Tentacles, Mr. Krabs, The Powerpuff Girls (Blossom, Bubbles, and Buttercup), Blythe Baxter, Zoe Trent, Pepper Clark, Penny Ling, Vinnie Terrio, Sunil Nevla, Minka Mark, Russell Ferguson, Gail Trent, Mitzi, Buttercream Sunday, Sugar Sprinkles, Youngmee Song, Sue Patterson, Jasper Jones, Sunset Shimmer, Judy Hopps, Nick Wilde, Belle, Ariel, Princess Jasmine, Princess Anna, and Elsa the Snow Queen guest star in this film.