Osteoarthritis (OA) is a common degenerative joint disease in older adults. A number of previous studies have demonstrated that natural flavonoids can serve as promising therapeutic drugs for OA. Kaempferol, a phytochemical ingredient mainly present in various fruits, has exhibited its prominent anti-inflammatory and antioxidant effects in numerous diseases. However, whether Kaempferol ameliorates the deterioration of arthritis remains to be elucidated. The aim of the present study was to investigate the therapeutic role of Kaempferol on OA in rat chondrocytes. The results revealed that Kaempferol significantly inhibited the interleukin (IL)-1 beta-induced protein expression of inflammatory mediators such as inducible nitric oxide synthase and cydo-oxygenase-2. In addition, the common matrix degrading enzymes [matrix metalloproteinase (MMP)-1, MMP-3, MMP-13 and a disintegrin and metalloproteinase with thrombospondin motif-5] induced by IL-1 beta were also suppressed by Kaempferol, and consequently abolished the degradation of collagen II. Furthermore, the anti-inflammatory effect of Kaempferol was mediated by the inhibition of the mitogen activated protein kinase-associated extracellular signal-regulated kinase and P38 signaling pathways. These results collectively indicated that Kaempferol can potentially prevent OA development and serve as a novel pharmacological target in the treatment of OA.