Neuropathic pain (NP) is a type of chronic pain affecting 6-8% of human health as no effective drug exists. The purinergic 2X4 receptor (P2X4R) is involved in NP. Neohesperidin (NH) is a dihydroflavonoside compound, which has anti-inflammatory and antioxidative properties. This study aimed to investigate whether NH has an effect on P2X4R-mediated NP induced by chronic constriction injury (CCI) of the sciatic nerve in rats. In this study, the CCI rat model was established to observe the changes of pain behaviors, P2X4R, and satellite glial cells (SGCs) activation in dorsal root ganglion (DRG) after NH treatment by using RT-PCR, immunofluorescence double labeling and Western blotting. Our results showed CCI rats had mechanical and thermal hyperalgesia with an increased level of P2X4R. Furthermore, SGCs were activated as indicated by increased expression of glial fibrillary acidic protein and increased tumor necrosis factor-alpha receptor 1and interleukin-1β. In addition, phosphorylated extracellular regulated protein kinases and interferon regulatory factor 5 in CCI rats increased. After NH treatment in CCI rats, the levels of above protein decreased, and the pain reduced. Overall, NH can markedly alleviate NP by reducing P2X4R expression and SGCs activation in DRG.