Objective - To determine whether glucosamine and chondroitin sulfate (CS) at concentrations approximating those achieved in plasma by oral administration would influence gene expression of selected mediators of osteoarthritis in cytokine-stimulated equine articular chondrocytes. Sample population - Samples of grossly normal articular cartilage obtained from the metacarpophalangeal joint of 13 horses. Procedure - Equine chondrocytes in pellet culture were stimulated with a subsaturating dose of recombinant equine interleukin (reIL)-1β. Effects of prior incubation with glucosamine (2.5 to 10.0 μg/mL) and CS (5.0 to 50.0 μg/mL) on gene expression of matrix metalloproteinase (MMP)-1, -2, -3, -9, and -13; aggrecanase 1 and 2; inducible nitric oxide synthase (iNOS); cyclooxygenase (COX)-2; nuclear factor κB; and c-Jun-N-terminal kinase (JNK) were assessed by use of a quantitative real-time polymerase chain reaction assay. Results - Glucosamine at a concentration of 10 μg/mL significantly reduced reIL-1β-induced mRNA expression of MMP-13, aggrecanase 1, and JNK. Reductions in cytokine-induced expression were also observed for iNOS and COX-2. Chondroitin sulfate had no effect on gene expression at the concentrations tested. Conclusions and clinical relevance - Concentrations of glucosamine similar to those achieved in plasma after oral administration in horses exerted pretranslational regulation of some mediators of osteoarthritis, an effect that may contribute to the cartilage-sparing properties of this aminomonosaccharide. Analysis of results of this study indicated that the influence of CS on pretranslational regulation of these selected genes is limited or lacking.