"We are saddened to report that during today’s rodeo, a steer sustained a severe injury that was untreatable and as a result was humanely euthanized," said the Stampede in a release.
It happened during the last round of the steer wrestling event in front of thousands of spectators.
The cowboy competitor immediately called for assistance after the steer failed to rise.
Rodeo workers surrounded the steer and competitor with a tarp before the animal was rushed off the field on a stretcher.
Three veterinarians on scene assessed the situation and determined the injury was not treatable.
A post-mortem will not be performed, as the veterinarians believe the injuries were self-evident and do not require further investigation.
Calgary Stampede officials say the run appeared to be normal, but independent livestock specialists will be reviewing the event video to determine if there were any contributing factors.
The Calgary Stampede says more than 1,000 steers have participated in the event over the past decade, and only one other animal has been fatally injured.
The Vancouver Humane Society called for the suspension of steer wrestling following the animal's death. The animal advocacy group has been a longtime critic of rodeo events at the Calgary Stampede.
Zane Hankel, the cowboy involved in the run, did not want to comment on the incident to CBC News. The Stampede's chief veterinarian is expected to hold a media briefing later this evening.