Directors Colin and Greg Strause, inspired by Terminator 2: Judgment Day, moved to Los Angeles and founded the visual effects company Hydraulx, which worked on films like The Day After Tomorrow, Poseidon and 300, helping build a strong relationship with 20th Century Fox that led to their hiring on this film.

The film's original title was Alien vs. Predator: Survival of the Fittest before it was changed.

The brothers had previously unsuccessfully pitched an idea for the first Alien vs. Predator film and also proposed a Wolfenstein film to Fox before being hired to direct this sequel in late spring 2006, with limited time before filming began that fall.

The directors wanted actor Adam Baldwin to reprise his role as Agent Garber from Predator 2, but were unable to secure him, so the role was replaced with a new character played by Robert Joy.

The lead Predator character was nicknamed "Wolf" by the production team after Harvey Keitel's character in Pulp Fiction, since both characters serve as a 'cleaner'.

Filming began on September 23, 2006, in Vancouver, British Columbia, on a tight 52-day shooting schedule.

During filming breaks, the Strause brothers simultaneously supervised visual effects work on 300, Shooter and Fantastic Four: Rise of the Silver Surfer using an in-house dailies tracking system called Mavis and Lucy.

Hydraulx produced about 460 of the film's 500 visual effects shots, including a nuclear explosion created using Maya fluids and BA Volume Shader; the visual effects team peaked at 110 people, nearly the entire Hydraulx staff.

The interior of the Predator ship was created entirely with CGI rather than a physical set because the directors felt it would be more cost effective; aside from exterior spaceship shots, the filmmakers tried to shoot everything else in-camera.

The song "Wach auf!" ("Wake Up") by German band Oomph! was released as a promotional single for the film in January 2008, using clips from the movie in its music video, despite not appearing on the official CD soundtrack.

Later Predator franchise film The Predator features references to this movie, including shurikens and a spear made from an Alien tail, similar to the one Scar creates in the first Alien vs. Predator film.

Actress Françoise Yip reprised her role as Ms. Yutani from this film in The Predator, though most of her role was cut from the final version aside from silent background appearances.