The ethanol extracts of Puerariae Radix inhibited cyclooxygenase-2 (COX-2) activity in bone marrow derived mast cells (BMMC). COX-2 is responsible for the production of large amounts of proinflammatory prostaglandins (PGs) at the inflammatory site. We have investigated the anti-inflammatory effect of ethyl acetate fraction from ethanol extract of Puerariae Radix (EPR), and attempted acetic acid induced writhing to verify the analgesic effect. Inflammation was induced by interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a), and lipopolysaccharide (LPS). EPR showed strong inhibitory efficacy against cytokine-induced proteoglycan degradation, prostaglandin production, nitric oxide (NO) production, and matrix-metalloproteinases (MMPs) expression in mouse macrophage and rabbit articular chondrocyte. In the writhing test, EPR exhibited a dose-dependent inhibition of writhing. The results indicate that EPR have anti-inflammatory and analgesic activities, and could be a good herbal medicine candidate for treating of osteoarthritis (OA).