Objective To assess the relationship between peripheral TRPV1 and P2X3 in normal healthy rats toelucidate the regulation mechanism of peripheral pain sensation. Methods Healthy male SD rats were randomly dividedinto a control group, TRPV1 agonist group, P2X3 agonist group, TRPV1 agonist + P2X3 agonist group, TPRV1 agonist +P2X3 inhibitor group, and P2X3 agonist +TRPV1 inhibitor group. Within 20 min after subcutaneous and intraplantarinjections of TRPV1 or P2X3 agonist and/ or inhibitor, the number of foot contractions and the duration of leg raising/licking in each group were measured, respectively. The expression and co-expression of positive cells of L4DRG TRPV1and P2X3 were observed using immunofluorescence. The correlation between TRPV1 and P2X3 at L4 DRG levels wasobserved using immunoprecipitation. Results The P2X3 agonist did not alleviate pain behavior induced by the TRPV1agonist, and the P2X3 inhibitor did. The TRPV1 agonist increased pain behavior induced by P2X3 agonist, and the TRPV1inhibitor did not reduce pain behavior induced by P2X3 agonist. The P2X3 agonist increased the positive area expression ofTRPV1 in at the L4DRG level, and the TRPV1 agonist increased the positive area expression of P2X3 at the L4DRG level;TRPV1 and P2X3 were co-expressed and co-precipitated at the L4 DRG level. Conclusions There is an interactionbetween TRPV1 and P2X3 at the peripheral neuron level, whereby they promote each other’s expression. When one is inhibited, the other is reduced in function.