Nicotine withdrawal (NTW) has been shown to increase pain sensitivity. However, the pathogenesis of NTW-induced hyperalgesia syndrome is unknown. Microglial activation, with increased expression of the P2X4 receptor (P2X4R) and brain-derived neurotrophic factor (BDNF) as important markers, is associated with hyperalgesia; therefore, these markers may represent an unprecedented target to prevent hyperalgesia. In this study, we explored the contributions of spinal microglial P2X4R–BDNF signaling in NTW-induced hyperalgesia. Immunohistochemical analysis showed that spinal microglia were activated and that the P2X4R level was increased and colocalized with ionized calcium-binding adapter molecule 1 in NTW-induced hyperalgesia. Furthermore, we showed that microglial activation with NTW resulted in an increased expression of spinal P2X4R and an elevated release of BDNF. Intrathecal minocycline (a specific inhibitor of microglial activation) reversed thermal hyperalgesia as well as increased the spinal microglial P2X4R and BDNF levels induced by NTW. To the best of our knowledge, the present study provides evidence that spinal microglial P2X4R–BDNF signaling is critical for the development of NTW-induced hyperalgesia.