Lammtarra, who won the 1995 Derby in a record time, has died at the age of 22.
The Gainsborough homebred, who also triumphed in the King George and the Arc, was put to sleep after a short illness.
He is one of only two horses to win all three races, along with Mill Reef.
"Lammtarra was very intelligent and a true gentleman," said stud director Liam O'Rourke. "He will be missed by everyone."
Sired by the legendary Derby winner Nijinsky, Lammtarra was voted the Cartier Three-Year-Old European Champion Colt in 1995.
His victory in the Derby was all the more remarkable as it came on his seasonal debut.