The effects of puerarin on acute nociception mediated by the P2X3 receptor in rat dorsal root ganglia (DRG) were studied. Intrathecal injection of adenosine triphosphate (ATP) or α,β-methylene-ATP (α,β-meATP) could potentiate acute nociceptive responses induced by intraplantar injection of ATP or α,β-meATP in rat hindpaw. Intraplantar injection of formalin increased the expression of P2X3 in rat DRG and induced acute nociceptive responses. These induced nociceptive responses were inhibited by intrathecal injection of puerarin, which also decreased the up-regulated expression of P2X3 mRNA and protein in DRG induced by intraplantar injection of formalin. Molecular docking studies revealed that puerarin could interact perfectly with homology-modeled rat P2X3 receptor (rP2X3). rP2X3 can be restricted to binding ATP because of its combination with puerarin (increasing the concentration of free ATP), therefore blocking the rP2X3 channel. Whole cell patch clamp recording showed that puerarin inhibited the potentiation of P2X3 receptor-mediated currents induced by lipopolysaccharide. These results demonstrated that puerarin inhibited nociceptive responses induced by ATP, α,β-meATP, or formalin and its inhibitory effect was mediated by reduction of inflammatory pain-induced up-regulation of P2X3 receptor expression and blockade of ATP binding sites on the P2X3 receptor. Our results suggest that puerarin could decrease acute pain mediated by the P2X3 receptor in the DRG. Key words: Puerarin, acute nociceptive response, P2X3 receptor, dorsal root ganglia, whole cell patch clamp.