Despite several lines of evidence indicate that spinal microglia participate in pain processing, it is pivotal to know whether there are differential roles of microglia in pain hypersensitivity between male and female mice. Induction of Gq-DREADD in spinal microglia elicited mechanical allodynia in male mice, but not in female mice, through the upregulation of inflammatory molecules. Peripheral nerve injury-induced mechanical allodynia was suppressed by the treatment of PLX3397, a microglial inhibitor, in male but not in female mice. Importantly, the effects of microglial activators and inhibitors (i.e., Gq-DREADD and PLX3397) in the spinal dorsal horn of male mice was significantly greater than that of female mice. Moreover, nerve injury-induced mechanical allodynia in female mice was prevented by administration of testosterone propionate, suggesting that androgens might determines sex differences of microglia. Collectively, male microglia have a potential to exacerbate pain sensitivity in mice, and further study on androgen actions may provide the key mechanisms underlying sex differences in spinal microglia.