Mangiferin is a kind of polyphenol chemical compound separated from these herbal medicines of Mangifera indica L., Anemarrhena asphodeloides Bge. and Belamcanda chinensis L., which has anti-inflammatory, anti-virus, and other physiological activities without toxic effects. Osteoarthritis (OA) is a chronic disease that is also a kind of arthritis disease in which articular cartilage or bones under the joint is damaged. In addition, artificial replacements are required in severe cases. At present, there are not too much researches on the potential biological activities of mangiferin that plays a protective role in the treatment of OA. In this study, we evaluated the protective effect of mangiferin on osteoarthritis (OA) in vitro and in vivo. First, the effect of different concentrations of mangiferin on rat chondrocytes was determined by MTT assay. Second, the effects of mangiferin on the expression levels of matrix metalloproteinase (MMP)-13, TNF-α, Collagen II, Caspase-3, and cystatin-C in interleukin-1β (IL-1β)-induced rat chondrocytes were examined by the real-time polymerase chain reaction in vitro, meanwhile the effects of mangiferin on the nuclear factor kappa-B (NF-κB) signaling pathway were also investigated by Western Blot. Finally, the antiosteoarthritic protective effect of mangiferin was evaluated in the rat model by anterior cruciate ligament transection (ACLT) combined with bilateral ovariectomy-induced OA in vivo. The results showed that the mangiferin was found to inhibit the expression of MMP-13, TNF-α, and Caspase-3 which also increased the expression of Collagen II and cystatin-C in IL-1β-induced rat chondrocytes. In addition, IL-1β-induced activation of nuclear factor kappa-B (NF-κB) and the degradation of inhibitor of κB (IκB)-α were suppressed by mangiferin. For the in vivo study in a rat model of OA, 100 μl of mangiferin was administered by intra-articular injections for rats, the results showed that the cartilage degradation was suppressed by mangiferin through Micro CT and Histological Examination. According to both in vitro and in vivo results, mangiferin has a protective effect in the treatment of OA which may be a promising therapeutic agent for OA.