Osteoarthritis (OA), in which inflammation plays a crucial role, is the most common joint disease characterized by cartilage degradation. Neferine (Nef), a dibenzyl isoquinoline alkaloid, has shown its anti-inflammatory effects on other inflammatory diseases. Therefore, we hypothesized that Nef might also have an anti-inflammatory effect on OA and explored its effect on IL-1 beta-treated rat chondrocytes. Sprague Dawley (SD) rat chondrocytes were stimulated with IL-1 beta (10 ng/ml) and Nef (1, 5, and 10 mu M) or IL-1 beta (10 ng/ml) alone for 24 h. Expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), matrix metalloproteinases (MMPs), and thrombospondin motifs-5 (ADAMTS5) was determined by quantitative real-time PCR and Western blotting. Expression of collagen II and aggrecan was examined by Western blotting, immunofluorescence, and safranin O staining. In addition, activation of MAPK and NF-kappa B signaling pathway was examined by Western blotting, and p65 nuclear translocation was evaluated by immunofluorescence. Nef reduced expression of inflammatory regulators (iNOS and COX-2) in IL-1 beta-treated chondrocytes. Expression of IL-1 beta-induced major catabolic enzymes (MMP3, MMP13, and ADAMTS5) was inhibited by Nef. Meanwhile, downregulation of collagen II and aggrecan expression was also ameliorated. Furthermore, Nef dampened abnormal activation of MAPK and NF-kappa B signaling pathway triggered by IL-1 beta. Overall, the results above showed that Nef inhibited IL-1 beta-induced excess production of inflammatory and catabolic factors in rat chondrocytes via inhibiting the MAPK and NF-kappa B pathways, suggesting a promising pharmacotherapy for OA.