Eight Belles was dead just a few minutes after the favourite Big Brown won a convincing race by more than four lengths on the Churchill Downs track.
But the eyes of thousands of fans were on the plight of Eight Belles, who had collapsed on the track after the finish. She received a lethal injection after ambulances were moved into position around the stricken horse.
A writer for the national USA Today newspaper asked: "Can we have a big-time race day without seeing a valiant four-legged competitor crippled and condemned to death?"
Eight Belles was the only filly in the 20-strong weekend field.
Eight Belles' death comes with the memory of Kentucky Derby winner Barbaro fresh in the minds of many racing followers.
Barbaro won the Kentucky Derby in 2006. He shattered a rear hind leg at the start of the Preakness - the second leg of the Triple Crown - and was euthanized eight months later as a result of laminitic complications.
Animal welfare group PETA, commenting on the death of Eight Belles, slammed what it called the "sordid truth" about racing.
"With the shock of Barbaro's death at the 2006 Preakness still fresh in their minds, the Kentucky Derby crowd saw for themselves the sordid truth about what racing means for the horses involved," it said of Eight Belles' death.
"This young filly's death may have been humane, but the race sure wasn't."
It was unhappy over the jockey's use of the whip down the final stretch.
"While the trainers, jockeys, and owners may weep their crocodile tears today over Eight Belles' euthanasia, they will be back on the track tomorrow, putting other horses at risk.
"Thoroughbreds are raced on hard dirt surfaces - like the one at Churchill Downs. Their bones simply can't take it, as Eight Belles' two broken front legs showed last night.
"Despite the wealth associated with thoroughbred racing, for the horses - most of whom end up broken, cast off, or sent to Europe to be killed for the dinner table - it's a dirty business and no better than dogfighting."