Last year's Derby runner-up The Cliffsofmoher suffered a fatal injury in the Melbourne Cup won by Cross Counter.
Cross Counter became the first British-trained horse to triumph in the 158th running of Australia's famous race.
But The Cliffsofmoher, trained in Ireland by Aidan O'Brien, was put down after sustaining a fractured shoulder.
The five-year-old is the fifth horse in the last six years to die after being injured in the two-mile race.
Jockey Ryan Moore was not injured.
"It is with sadness that we confirm The Cliffsofmoher had to be humanely euthanised after sustaining a fractured right shoulder during the running of the Melbourne Cup at Flemington," said Racing Victoria spokesman Jamie Stier.
"The horse received immediate veterinary care. However, it was unable to be saved due to the nature of the injury sustained.
"This was an unfortunate incident that happens infrequently, with Victoria having one of the best safety records in world racing."
But RSPCA Australia said it was concerned about the welfare of racehorses.
"Our thoughts are with animal lovers across the country who have been affected by this loss, and confronted by the very real risks posed by racing to the horses involved," said spokesperson Jane Speechley.
"The racing industry has much work to do across industry standards and enforceable regulations, to meet community expectations and care for the welfare of the animals involved."
The Cliffsofmoher, whose name was changed from Cliffs Of Moher to race in Australia, was runner-up to stablemate Wings Of Eagles in the 2017 Derby at Epsom.
He had finished third last month in the Caulfield Cup in Australia for the 21-times Irish champion trainer O'Brien.
- Australian Hugh Bowman, the rider of runner-up Marmelo in the Melbourne Cup, was given a 35-meeting ban for careless riding, excessive whip use and weighing in overweight. Five other jockeys, including winning rider Kerrin McEvoy (A$3,000/£1,650) were fined for breaching the whip rules.