According to Nimród Antal and Robert Rodriguez on the commentary, in the script, the character Cuchillo was described as "a guy who looks like Danny Trejo." When Danny Trejo heard this, he called Robert Rodriguez and said, "hey, I heard there's a guy in the script for 'Predators' who looks just like Danny Trejo, and guess what, I look just like Danny Trejo!"
The alien creature that pursues Edwin (Topher Grace) through the jungle, during the ambush scene, is a slightly modified version of the original, abandoned Predator design from 1987.
The "hunting dogs" are based on one of the creature skulls seen in the trophy case on-board the Predator ship, near the end of Predator 2 (1990).
Hanzo is missing two fingers on his left hand. The reason that the Yakuza traditionally cut their fingers starting from the left pinkie, is that these fingers have a vital role in controlling a Japanese sword, and their loss would significantly impair a duelist. Hanzo must be a formidable swordsman to be able fight in close combat as he did, despite the loss of his fingers.
The film's basic plot was conceived in 1994, when Robert Rodriguez was working on Desperado (1995). He presented a draft of the script to 20th Century Fox, but they turned it down, because the budget required was too large. Fifteen years later, the studio decided to follow through with his script; in the end, an updated version of his script.