Neuropathic pain can arise from nerve injury caused by disease, infection or trauma. It is among the most debilitating chronic pain conditions and is refractory to the current available pharmacological treatments. Converging evidence suggests that ATP-gated P2X4Rs on spinal microglia are implicated in the development and maintenance of neuropathic pain. P2X4Rs are upregulated in the spinal dorsal horn following nerve injury, and stimulation of P2X4Rs drives p38-MAPK activation and BDNF release, which leads to aberrant nociceptive processing in the spinal cord. However, recent evidence suggests that the role of microglia in neuropathic pain is sexually dimorphic. In this study, I provide evidence that microglial P2X4Rs are a key cellular point of sexual divergence in the development of pain hypersensitivity following PNI. In cell cultures, I determined that ATP-stimulation upregulated P2X4Rs leading to p38-MAPK activation and SNARE-dependent release of BDNF only in microglia isolated from male postnatal rats. Additionally, I found that intrathecal injection of P2X4R-stimulated microglia derived from males, but not from females, induced mechanical allodynia in non-injured naïve rodents. I also show that P2X4R expression and function are increased in spinal microglia isolated from only male rats following PNI. Finally, my data provides novel evidence that transcription factor IRF5 differentially regulates P2rx4 gene expression in males and females, providing a possible explanation for the dimorphisms underlying P2X4R-signalling. Together, the findings of this study suggest that the microglial P2X4R is a critical point of sexual divergence in pain hypersensitivity, and may provide a cellular basis for the development of more effective, sex-dependent treatment options for neuropathic pain.