To evaluate the role of ghrelin on diabetic neuropathic pain and its effect on the expression of purinergic receptor 2X-4/NOD-like receptor-3 (P2X4/NLRP3). Methods: Forty male Sprague-Dawley rats were randomly allocated into 5 groups (n=8 each): normal + ghrelin group (group NG), normal + [D-Lys3]-GHRP-6 group (group ND), diabetes group (group D), diabetes + ghrelin group (group DG), and diabetes + ghrelin + [D-Lys3]-GHRP-6 group (group DGD). [D-Lys3]-GHRP-6 served as a ghrelin-specific inhibitor. Group NG received intraperitoneal injections of 200 μg/kg ghrelin for 6 weeks, and group ND received intraperitoneal injections of 50 mg/kg [D-Lys3]-GHRP-6 for 6 weeks. Diabetes mellitus was induced by intraperitoneal injections of 60 mg/kg 1% streptozotocin. Three days after establishment of diabetes model, group DG was intraperitoneally given 200 μg/kg ghrelin and group DGD given 200 μg/kg ghrelin plus 50 mg/kg [D-Lys3]-GHRP-6 for 6 weeks. Mechanical withdrawal threshold (MWT) and motor nerve conduction velocity (MNCV) were measured at 2, 4, and 6 weeks. After 6 weeks the rats were sacrificed. Rat spinal cords were examined for pathology change by Nissl staining and pathology change of the sural nerve by electron microscopy (EM). Expression of ghrelin, P2X4, NLRP3, and IL-1β in the spinal cord was detected by western blot. Results: Compared with group NG, group ND spinal Nissl staining and sural nerve EM showed normal spinal cell structure; there was no significant change in MWT and MNCV and no significant difference in the expression of P2X4, NLRP3, and IL-1β (P>0.05). Spinal Nissl staining and sural nerve EM of group D showed partial cellular damage, decreased MWT and MNCV, down-regulated expression of ghrelin, and up-regulated expression of P2X4, NLRP3, and IL-1 β (P<0.05). Compared with group D, group DG showed normal spinal cell structure, increased MWT and MNCV, up-regulated expression of ghrelin, and down-regulated expression of P2X4, NLRP3, and IL-1 β (P<0.05). Compared with group DG, group DGD showed severe cell structure damage, decreased MWT and MNCV, down-regulated expression of ghrelin, and up-regulated expression of P2X4, NLRP3, and IL-1 β (P<0.05). Conclusion: Ghrelin may be involved in diabetic neuropathic pain through the P2X4/NLRP3 signaling pathway.