Summary Objective Glucosamine (GLN) and chondroitin sulfate (CS) are widely used to alleviate symptoms of osteoarthritis (OA). However, the mechanism(s) of action of these nutraceuticals remains unresolved. In the present study, we determined the effect of physiologically relevant concentrations of GLN and CS on gene expression and synthesis of nitric oxide (NO) and prostaglandin E 2 (PGE 2) in cytokine-stimulated articular cartilage explants. Methods Using bovine articular cartilage explants in culture stimulated with IL-1, the effects of physiologically relevant concentrations of GLN and CS on gene expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGEs1) were assessed with quantitative real-time polymerase chain reaction (Q-RT-PCR). The production of NO and PGE 2 was also quantified. Results CS and the GLN and CS combination at concentrations attainable in the blood down-regulated IL-1 induced mRNA expression of iNOS at 24 and 48 h post-culture. Up-regulated iNOS expression at 24 h by IL-1 was also suppressed by GLN. GLN and CS transiently repressed the cytokine-stimulated mPGEs1 transcript. Synthesis of NO was reduced with CS alone and the combination after 24 h of culture. Repression of COX-2 transcripts by GLN and CS was accompanied by concomitant reduction in PGE 2. Conclusion Our results indicate that physiologically relevant concentrations of GLN and CS can regulate gene expression and synthesis of NO and PGE 2, providing a plausible explanation for their purported anti-inflammatory properties.