Neuropathic pain (NP) is a serious problem caused by lesions or diseases in the peripheral or central nervous system. It has been widely known that various immune cells contribute to the development and maintenance of NP, and thus the importance of spinal microglia in pain formation. To more specifically evaluate the role of microglia on pain regulation and its sex-dependency, we used Gi or Gq-Designer Receptors Exclusively Activated by Designer Drugs（Gi/Gq-DREADD）system driven by microglia-specific cx3cr1 promoter in both male and female mice (indicated as CX3CR1-hM4Di or CX3CR1-hM3Dq mice, respectively). In CX3CR1-hM4Di or CX3CR1-hM3Dq mice, expression of hM4Di or hM3Dq was detected in Iba1-positive microglia, but not in astrocytes or neurons. Intraperitoneal (i.p.) or intrathecal (i.t.) administration of clozapine-N-oxide (CNO), a ligand for hM4Di and hM3Dq, attenuated mechanical allodynia, evaluated by von Frey test, in CX3CR1-hM4Di mouse models of NP. While, either i.p. or i.t. administration of CNO induced mechanical allodynia in CX3CR1-hM3Dq mice. More importantly, these effects on pain regulation in CX3CR1-hM4Di and CX3CR1-hM3Dq mice were only observed in male, but not in female mice. These findings support the notion that sex-dependent pain-regulatory effects of spinal microglia in mice.