Cordycepin is widely used as for its various pharmacological activities, such as anti-inflammation, anti-angiogenesis, anti-aging, anti-tumor and anti-proliferation. However, the precise role of cordycepin on chondrocytes is not clear. In the present study, we examined the inhibitory effects of cordycepin on interleukin-1 beta (IL-1β)-induced glycosaminoglycan (GAG) release, nitric oxide production as well as gene expressions of inflammatory and catabolic mediators in human cartilage and chondrocytes. Cartilage explants and human chondrocytes were cultured in the absence or in the presence of IL-1β (10 ng/ml) and with or without cordycepin (5-100 μM). GAG content in the cartilage explants was measured by using the dimethylmethylene blue method and Safranin O staining. Nitric oxide level was determined by Griess reaction. Expressions of MMP-1, MMP-13, cathepsin K, cathepsin S, ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs-4) and ADAMTS-5, inducible nitric oxide synthase (iNOS) and cyclooxgenase-2 (COX-2) were evaluated by real-time quantitative PCR. We found that cordycepin suppressed IL-1β-stimulated GAG release. Gene expressions of catabolic enzymes, including MMP-1, MMP-13, cathepsin K, cathepsin S, ADAMTS-4 and ADAMTS-5, were decreased by cordycepin in a dose-dependent manner. In addition, cordycepin inhibited IL-1β-induced COX-2 and iNOS expression at the transcript level as well as blocked NO production. Our results suggest that cordycepin may possess chondroprotective effect by preventing cartilage denegation and interfering inflammatory response in the pathogenesis of OA.