Bob Baffert, a Hall of Fame trainer, announced in a press conference at Churchill Downs Sunday that the horse failed a post-race drug test. Churchill Downs suspended Baffert as a result.
Medina Spirit was found to have 21 picograms of the steroid betamethasone, which is double the legal limit in Kentucky racing, Baffert said.
On May 1, Medina Spirit won the Kentucky Derby by a half-length. Baffert's team learned of the positive result from Kentucky officials on Saturday, he said.
The test results were obtained by the Kentucky Horse Racing Commission on Friday, Marc Guilfoil, executive director of the commission, said in a statement.
The win gave Baffert his seventh victory, a record for the race.
He described the test result as a "complete injustice," saying Medina Spirit had never been treated with betamethasone, a joint injection/anti-inflammatory medication, and that he did not know how the horse could have tested positive.
"I got the biggest gut-punch in racing, for something I didn't do," he said.
Medina Spirit has not been officially disqualified, but that could still happen once more tests are complete, Baffert said.
If the findings of the investigation are upheld, Medina Spirit's Kentucky Derby win will be invalidated, according to a statement from Churchill Downs.
"Failure to comply with the rules and medication protocols jeopardizes the safety of the horses and jockeys, the integrity of our sport and the reputation of the Kentucky Derby and all who participate," the statement read. "Churchill Downs will not tolerate it."
Baffert vowed to be transparent throughout the investigation but added that his team will conduct their own investigation.
"During the investigation, both the trainer and owner of the horse will be afforded due process, and opportunity to appeal,"Guilfoil said in a statement, declining to comment further.
Baffert is preparing to race Medina Spirit at the Preakness Stakes in Baltimore on Saturday.
Baffert had been suspended by the Arkansas Racing Commission for 15 days after two of his horses that won at Oaklawn Park tested positive for the painkiller lidocaine, The Associated Press reported.
He won an appeals case last month after testifying that the horses had been exposed to the drug inadvertently.
ABC News' Joshua Hoyos and Ben Stein contributed to this report.