The 16-year-old horse, named Ezzy, had to be euthanized after it tore a ligament in a front leg during the first heat of Sunday night's competition to cap the annual Rangeland Derby.
The animal was an outrider horse that did not make contact with any other horses or wagons.
Veterinarians at the scene could not repair the injury, which they say is common in racing and other types of horses.
Another outrider horse had to be euthanized on Saturday after it suffered a similar type of injury.
Two others that were harnessed to chuckwagons were put down after track-related accidents earlier in the week.
"(Ezzy) was examined before the Stampede as well as each night before it raced and it was examined tonight and it was examined before the race track, so there is no indication this was going to happen," said Greg Evans, a veterinarian with the Stampede. "Even in clinically sound horses, it can occur during racing."
Dave Galloway, who owned Ezzy, said everyone on his team loved the horse.
"He was calm, he was our fastest horse. Everyone called him Fuzzy Ezzy. I know it's pretty sombre back at our barn right now and we spend our lives taking care of the lives of these horses, showcasing them and what they can do. Racing for Ezzy was effortless."
Kurt Kadatz, communications director for the Stampede, said the organization has made advances in animal fitness and welfare, but it's not proud of its safety record this year.
"We are really going to look hard and find out what we can be doing differently to achieve a better result because we certainly do take it very seriously. Certainly you can see, by these numbers we are not happy."
Kadatz said he believed one horse died in 2013 and one last year.
Animal rights groups have been scrutinizing the Stampede's safety measures, saying they are not enough to prevent animal deaths.
An estimated 1.2 million people attended this year's Stampede.
(CHQR, CTV Calgary)
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