Red Cadeaux, which was runner-up at the Melbourne Cup three times, has been put down because of injuries it suffered in this year's cup.
The gelding pulled up in the final straight of the race three weeks ago, and it was later confirmed he had broken his left foreleg.
Emergency surgery was performed but his trainer says there there were "irreversible" complications.
Red Cadeaux became a fan favourite in Australia after brave runs to finish second at the Melbourne Cup in 2011, 2013 and 2014.
Overall, he won won seven and finished second in 13 of his 54 races.
Racing Victoria says few horses have endeared themselves to the Australian public like Red Cadeaux.