Edgar Prado, who used the red-and-white leather saddle to guide Barbaro to a dominating victory in last year's Derby, also used it while winning the 2002 and 2004 Belmont Stakes.
The saddle will be auctioned off at the Mint Jubilee Gala on May 4, the night before this year's Derby.
"Barbaro was one of the greatest horses to ever run the Derby," Prado said. "I only used the saddle for major races."
Prado was aboard Barbaro during last year's Preakness Stakes when the 3-year-old colt broke down in the front stretch after injuring his right hind leg.
Prado's ability to quickly pull up and calm the horse helped doctors immediately address Barbaro's injuries. Barbaro underwent emergency surgery after the race and spent several months in rehabilitation before being euthanized in January following complications from the injury.
All proceeds from the sale of the saddle will go to the Permanently Disabled Jockeys Fund, a division of National Thoroughbred Racing Association Charities. There are 58 severely injured jockeys receiving vital assistance each month from the fund.