AbstractThe pro‐inflammatory phenotype of microglia usually induces neuroinflammatory reactions in neuropathic pain. Glycometabolism shift to glycolysis can promote the pro‐inflammatory phenotype transition of microglia. The omics data analysis suggest a critical role for Lyn dysregulation in neuropathic pain. The present study aimed at exploring the mechanism of Lyn‐mediated glycolysis enhancement of microglia in neuropathic pain. Neuropathic pain model was established by chronic constriction injury (CCI), then pain thresholds and Lyn expression were measured. Lyn inhibitor Bafetinib and siRNA‐lyn knockdown were administrated intrathecally to evaluate the effects of Lyn on pain thresholds, glycolysis and interferon regulatory factor 5 (IRF5) nuclear translocation of microglia in vivo and in vitro. ChIP was carried out to observe the binding of transcription factors SP1, PU.1 to glycolytic gene promoters by IRF5 knockdown. Finally, the relationship between glycolysis and pro‐inflammatory phenotype transition of microglia was evaluated. CCI led to the upregulation of Lyn expression and glycolysis enhancement in microglia of spinal dorsal horn. Bafetinib or siRNA‐lyn knockdown intrathecally alleviated pain hyperalgesia, suppressed glycolysis enhancement and inhibited nuclear translocation of IRF5 in CCI mice. Also, IRF5 promoted the binding of transcription factors SP1, PU.1 to glycolytic gene promoters, and then the enhanced glycolysis facilitated the proliferation and pro‐inflammatory phenotype transition of microglia and contributed to neuropathic pain. Lyn‐mediated glycolysis enhancement of microglia contributes to neuropathic pain through facilitating IRF5 nuclear translocation in spinal dorsal horn.