Several lines of evidence indicate that spinal microglia exacerbate abnormal pain processing. Recent findings demonstrated there are significant functional differences of microglia in pain hypersensitivity between male and female animals, but underlying mechanisms are poorly understood. Here, we investigated whether androgens affect sex differences of microglia in neuropathic pain model mice. Peripheral nerve injury-induced mechanical allodynia was suppressed by the treatment of PLX3397, a microglial inhibitor, in male but not in female mice, and the effects of PLX3397 in the spinal dorsal horn of male mice was significantly greater than that of female mice. Gonadectomy (GDX) decreased in serum testosterone concentration and mechanical pain threshold in male mice. Susceptibility of spinal microglia for PLX3397 in GDX-treated male mice was similar to that of normal female mice. Moreover, intrathecal administration of colony-stimulating factor 1 (CSF1) elicited mechanical allodynia in male mice, but not GDX-treated male mice or normal female mice. Collectively, functional roles of spinal microglia contributing pain hypersensitivity are different between male and female, and sex-dependent characters of spinal microglia might be determined through androgen actions.