Objective: To develop an in vitro model of cartilage injury in full-thickness equine cartilage specimens that can be used to simulate in vivo disease and evaluate treatment efficacy. Sample: 15 full-thickness cartilage explants from the trochlear ridges of the distal aspect of the femur from each of 6 adult horses that had died from reasons unrelated to the musculoskeletal system. Procedures: To simulate injury, cartilage explants were subjected to single-impact uniaxial compression to 50%, 60%, 70%, or 80% strain at a rate of 100% strain/s. Other explants were left uninjured (control specimens). All specimens underwent a culture process for 28 days and were subsequently evaluated histologically for characteristics of injury and early stages of osteoarthritis, including articular surface damage, chondrocyte cell death, focal cell loss, chondrocyte cluster formation, and loss of the extracellular matrix molecules aggrecan and types I and II collagen. Results: Compression to all degrees of strain induced some amount of pathological change typical of clinical osteoarthritis in horses; however, only compression to 60% strain induced significant changes morphologically and biochemically in the extracellular matrix. Conclusions and Clinical Relevance: The threshold strain necessary to model injury in full-thickness cartilage specimens from the trochlear ridges of the distal femur of adult horses was 60% strain at a rate of 100% strain/s. This in vitro model should facilitate study of pathophysiologic changes and therapeutic interventions for osteoarthritis.