We evaluated the effect of platelet-rich plasma-incorporated gelatin hydrogel microsphere (PRP/GM) together with subchondral drilling for cartilage regeneration in horses. Cartilage defects (diameter, 4.5 mm; depth, 3.0 mm) were created in both third carpal bones of six thoroughbred horses. A subchondral hole was drilled at the center of this cartilage defect (diameter, 2.0 mm; depth, 35 mm) in one randomly selected carpal joint (test joint), whereas no subchondral hole was made in the contralateral joint (control joint). Thereafter, the test joint was injected with PRP/GM and the control joint with saline-infused GM. Additional injections of PRP/GM were given 14 and 28 days after surgery. After 16 weeks, histological analysis was conducted. The macroscopic and histological scores of the test group were significantly higher than those of the control group (P < 0.05). These data indicated that the application of PRP/GM together with subchondral drilling accelerated cartilage regeneration in horses.