Objective To investigate the analgesic effect of bufalin and its underlying mechanisms. Methods The analgesic effect of bufalin was studied by the acetic acid-induced writhing response and hot plate test. The contents of NO and the activation of MAPK/p38/iNOS pathway in LPS-induced RAW264.3 were measured by Griess assay and Western blotting, respectively. Results Bufalin (0.5, 1.0 mg/kg) showed a significant inhibition of acetic acid-induced writhing response and prolonging of licking time in hot plate test at a dose-dependent manner (P<0.05). Bufalin (0.04, 0.08 μM) significantly reduced the contents of NO and inhibited the activation of MAPK/p38/iNOS in RAW264.7 cells. Conclusions The analgesic of bufalin is related to suppressing the activation of MAPK/p38/iNOS pathway and subsequently reducing the synthesis and releases of excessive NO.