Effective prevention and treatment of osteoarthritis for horses is still needed. This research tests the ability of glucosamine and chondroitin sulfate (GLN/CS) to mitigate inflammatory and mechanical stress in vitro. In this study, GLN/CS mediate this effect by a decrease of the synthesis of nitric oxide (NO) and a decrease of proteoglycan release from the extracellular matrix in stressed cartilage explants. Explants were cultured with interleukin-1 (IL-1) + mechanical trauma with and without GLN/CS. NO and prostaglandin E2 were measured as indicators of an inflammatory response. Glycosaminoglycans were measured as an indicator of cartilage breakdown. NO levels in the stressed explants with GLN/CS treatment were lower than the IL-1 + mechanical impact treatment alone and did not differ from control group. The glycosaminoglycan release was also lower in the GLN/CS treatment than the IL-1 + mechanical impact treatment, although the prostaglandin E2 concentration was not affected. This study offers some evidence that GLN/CS treatment can partially mitigate the catabolic response to inflammatory stress and mechanical trauma in equine cartilage explants. These results provide additional support for the continued study on the benefit of GLN/CS for horses with cartilage degeneration.