Many Clouds, winner of the Grand National in 2015, has collapsed and died after lowering the colours of Thistlecrack in an upset result in the Cotswold Chase at Cheltenham.
Having answered every call for jockey Leighton Aspell, the 8-1 winner, who got home by a head, collapsed after the winning post, being treated immediately by veterinary staff before his death was confirmed.
Trainer Oliver Sherwood paid tribute to the "horse of a lifetime".
"We've got to look forward and not look back. He's been the horse of a lifetime and I always said he would die for you and he's died for me and the team today doing what he does best," Sherwood said.
"He wanted to win that race, he was beaten and then fought back in the last 50 yards to win.
"I've been in the game 32 years and horses like that don't come along very often. It is going to very tough having an empty box there tomorrow and on Monday.
"We've got to be philosophical and celebrate the Hennessy and National wins and that was almost a career-best performance.
"He had wobbled after races before and people queried why he raced on, but what do you do with a horse like that? I thought, hand on heart, having had a wind op that he might have been struggling for oxygen and hence the reason we did it."
British Horseracing Authority media manager Robin Mounsey said it was a rare incident which would be investigated.
"The care available for racehorses throughout their lives, and in particular on the racecourse, is first class," he said.
"All horses are observed by a vet before taking part in a race. After the race, Many Clouds was immediately tended to by Cheltenham's team of expert racecourse vets within seconds of the first sign that he was in any difficulty.
"Despite this treatment, sadly, the horse could not be saved.
"We cannot yet be certain of the cause of the incident, but steps will be taken to understand exactly what has happened. Incidents such as this are exceptionally rare."