This study investigated whether the neuronal P2X3 receptor in rat dorsal root ganglia (DRG) mediated the effects of hesperidin on neuropathic pain. The chronic constriction injury (CCI) model was used as a model of neuropathic pain. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured. The mRNA and protein expression levels were assayed by real-time RT-PCR and Western blotting. The results showed that mechanical and thermal hyperalgesia in the CCI rats were increased as compared to those in the sham group. The expression levels of P2X3 mRNA and protein in CCI rats were higher than those in the sham group. Dual-labelling immunofluorescence showed that the elevated P2X3 receptor was co-expressed with the neuronal marker NeuN in the DRG of CCI rats. Hesperidin treatment decreased both the mechanical and thermal hyperalgesia, and upregulated P2X3 expression in the CCI rats. Hesperidin treatment also reduced the ERK1/2 phosphorylation in the DRG of CCI rats. Moreover, hesperidin inhibited the P2X3 agonist ATP-induced currents in HEK293 cells transfected with the P2X3 plasmid. Therefore, hesperidin treatment could reverse the elevated expression of neuronal P2X3 receptor and reduce the activation of ERK1/2 in the DRG of CCI rats. Our findings suggested that hesperidin inhibited the nociceptive transmission mediated by the P2X3 receptor in neurons of DRG, and thus, relieved the mechanical and thermal hyperalgesia in CCI rats.