Goldikova failed in her bid for a fourth Breeders' Cup Mile as outsider Court Vision won at Churchill Downs.
The six-year-old French mare had won the last three runnings of the race but was beaten by the 64-1 shot.
Court Vision beat Turallure in a photo finish, while Goldikova survived an inquiry to claim third place.
She will now be retired and trainer Freddie Head said: "I think she has earned her place as one of the great horses of the last century."
Goldikova had gone off as 5-4 favourite for the race, but jockey Olivier Peslier was unable to guide her to victory, with the glory going to the Dale Romans-trained Court Vision, ridden by Robby Albarado.
Head added: "For two strides Olivier thought she was going to win again, but then she was gone.
"But it was the normal way to ride her. She has won a lot of her races by following the leaders and arriving late.
"We found excuses for her recent defeats, but maybe she has lost a bit of that special thing she had that made her so great. I think the mileage and years have taken their toll.
"Winning 14 Group One races and competing against the best is something exceptional.
"I'm very, very happy and proud at this moment, and I knew it was going to be tough for her.
"For myself, maybe the blues will come a little later."
Goldikova had been expected to face strong competition from Richard Hannon's Strong Suit, but he made little impact on the feature race of the Breeders' Cup meeting.
Jockey Richard Hughes said: "He got a bump on the first bend, and he was never quite happy after that.
"He seemed very conscious of the ground moving underneath him."