ATP is a ligand of P2X family purinoceptors, and exogenous ATP administration evokes pain behaviors. To date, there is a lack of systematic studies to address relationships between endogenous ATP and neuropathic pain. In this report, we took advantage of a mouse model of resiniferatoxin (RTX)-induced neuropathic pain to address the role of endogenous ATP in neuropathic pain. After RTX administration, endogenous ATP markedly increased in dorsal root ganglia (DRGs) (p < 0.01) and skin tissues (p < 0.001). The excessive endogenous ATP was removed by apyrase, an ATP hydrolyzing enzyme, administration via either a lumbar puncture route (p < 0.001) or an intraplantar injection (p < 0.001), which led to the normalization of neuropathic pain. In addition, intraplantar treatment with apyrase caused mechanical analgesia. Linear analyses showed that the densities of P2X3(+) neurons (r = -0.72, p < 0.0001) and P2X3(+) dermal nerves (r = -0.72, p < 0.0001) were inversely correlated with mechanical thresholds. Moreover, the contents of endogenous ATP in skin tissues were linearly correlated with P2X3(+) dermal nerves (r = 0.80, p < 0.0001) and mechanical thresholds (r = -0.80, p < 0.0001). In summary, this study demonstrated that enhanced purinergic signalling due to an increase in endogenous ATP after RTX-induced nerve injury contributed to the development of neuropathic pain. The data in this report provide a new therapeutic strategy for pain control by targeting the endogenous ligand of purinergic signalling.