Osteoarthritis (OA) in humans is associated with low circulating 25-hydroxyvitamin D3 [25(OH)D₃]. In vitamin D replete rats, radiolabeled 24R,25-dihydroxyvitamin D3 [24R,25(OH)₂D₃] accumulates in articular cartilage following injection of [³H]-25(OH)D₃. Previously, we showed that 24R,25(OH)₂D₃ blocks chondrocyte apoptosis via phospholipase D and p53, suggesting a role for 24R,25(OH)₂D₃ in maintaining cartilage health. We examined the ability of 24R,25(OH)₂D₃ to prevent degenerative changes in articular cartilage in an OA-like environment and the potential mechanisms involved. In vitro, rat articular chondrocytes were treated with IL-1β with and without 24R,25(OH)₂D₃ or 1α,25(OH)₂D₃. 24R,25(OH)₂D₃ but not 1α,25(OH)₂D₃ blocked the effects of IL-1β in a dose-dependent manner, and its effect was partially mediated through the TGF-β1 signaling pathway. In vivo, unilateral anterior cruciate ligament transections were performed in immunocompetent rats followed by intra-articular injections of 24R,25(OH)₂D₃ or vehicle (t = 0, 7, 14, 21 days). Tissues were harvested on day 28. Joints treated with vehicle had changes typical of OA whereas joints treated with 24R,25(OH)₂D₃ had less articular cartilage damage and levels of inflammatory mediators. These results indicate that 24R,25(OH)₂D₃ protects against OA, and suggest that it may be a therapeutic approach for preventing trauma-induced osteoarthritis.