Justify has been retired because of injury, connections of the undefeated American Triple Crown winner said in a statement.
The three-year-old son of Scat Daddy has been struggling with an ankle problem which has now brought the end to a magnificent career.
Trainer Bob Baffert said: "Justify had some filling in his ankle, and he is just not responding quick enough for a fall campaign.
"We all wanted to see Justify run again, but ultimately it is my responsibility to make sure he is perfect.
"Without 60-90 days, I can't be definite."
Justify is partly owned by WinStar Farm, whose president Elliott Walden said: "We all wanted him to finish his career in the Breeders' Cup Classic, but it was not meant to be.
"As has been reported, there is a possible sale to Coolmore in place, but that won't be finalised until a later date."
Justify retires with a perfect six-from-six record and has earned over €3.2m in prize-money.
Jockey Mike Smith, who partnered Baffert's star in five of his races, said: "There was never a time when I rode him that I felt like I was going to get beat.
"There was no horse who could run with him without sacrificing themselves, and there was no horse who could come get him.
"He truly is a gift from God."
