Red Cadeaux has been euthanised due to serious leg injury sustained at the Melbourne Cup earlier this month.
Racing Victoria made the announcement on Saturday after the 10-year-old triple Melbourne Cup runner-up suffered "irreversible complications" from surgery.
Red Cadeaux - pipped to the line in 2011, 2013 and 2014 - pulled up lame at Flemington on November 3, sustaining a fetlock injury during the famous race in Melbourne.
Trainer Ed Dunlop's initial fears about Red Cadeaux's welfare seemed to have been allayed in recent weeks but the crowd favourite's injuries were too severe to overcome.
"This is my saddest day in racing. Red Cadeaux was a much-loved member of our family and my thoughts are with his owner Mr [Ronnie] Arculli and my staff, particularly Robin Trevor-Jones and Steve Nicholson, who spent every day caring for this horse," Dunlop said in a statement.
"Unfortunately the complication was irreversible and the decision to euthanise the horse, whilst terribly hard, was made in his best interests.
"Red Cadeaux has given us and the racing public so much joy competing with great distinction across the world. He was an incredibly tough competitor with a wonderful nature and he will be dearly missed by all.
"I want to thank all the vets in Australia, particularly at the University of Melbourne Equine Clinic, that have worked tirelessly on the horse since the race and given him the very best of care.
"On behalf of Mr Arculli and my staff, I also want to thank the Australian public and his racing fans around the world for their wonderful support and well wishes during what has been an incredibly tough time for us all."