OBJECTIVE Cordycepin, a nucleoside derivative isolated from Cordyceps, has been reported to exert anti-inflammatory, antitumor, antidiabetic and renoprotective effects. Osteoarthritis (OA) is a degenerative joint disease with an inflammatory component that drives the degradation of cartilage extracellular matrix. This study aimed to assess the effects of cordycepin on human OA chondrocytes. METHODS In this study, human OA chondrocytes were pretreated with cordycepin at 10, 50 or 100 μM and subsequently stimulated with interleukin-1β (IL-1β) (5 ng/ml) for 24 h. Production of prostaglandin E2 (PGE2) and nitric oxide (NO) were evaluated by the Griess reaction and an enzyme-linked immunosorbent assay (ELISA). Gene expression of matrix metalloproteinase (MMP)-13, IL-6, inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) was measured by real-time polymerase chain reaction (PCR). MMP-13 and IL-6 proteins in culture medium were determined using cytokine-specific ELISA. Western immunoblotting was used to analyse the iNOS and COX-2 protein production in culture medium. Nuclear factor kappa-B (NF-κB) activity regulation was explored using Western immunoblotting. RESULTS Pretreatment with cordycepin significantly inhibited the production of PGE2 and NO induced by IL-1β. Cordycepin also significantly decreased the IL-1β-stimulated gene expression and production of MMP-13, IL-6, iNOS and COX-2 in OA chondrocytes. Pretreatment with cordycepin attenuated IL-1β-induced activation of NF-κB by suppressing degradation of its inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α) in the cytoplasm. CONCLUSIONS We show for the first time the anti-inflammatory activity of cordycepin in human OA chondrocytes. Thus, with this unique profile of actions, cordycepin may prove to be a potentially attractive and new therapeutic/preventive agent for OA.