The progressive increase in the prevalence of obesity in the population has resulted in increased healthcare costs and demands. Recent studies have revealed a positive correlation between pain and obesity, although the underlying mechanisms still remain unknown. Here, we aimed to clarify the role of microglia in altered pain behaviors induced by a high-fat diet (HFD) in male mice. We found that C57BL/6CR mice on a HFD exhibited spinal microglial reaction (increased cell number and up-regulated expression of p-p38 and CD16/32), increased tumor necrosis factor-α (TNF-α) mRNA and brain-derived neurotrophic factor (BDNF) protein as well as a polarization of spinal microglial toward a pro-inflammatory phenotype. Moreover, we found that when applied PLX3397 (a selective colony-stimulating factor-1 receptor (CSF1R) kinase inhibitor) to eliminate microglia of HFD-induced obesity mice, inflammation in the spinal cord was rescued, as were abnormal pain hypersensitivity. Intrathecal injection of Mac-1-saporin (a saporin-conjugated anti-mac1 antibody) resulted in decreased microglia and attenuated both mechanical allodynia and thermal hyperalgesia in HFD-fed mice. These results indicate that the pro-inflammatory functions of spinal microglia have a special relevance to abnormal pain hypersensitivity in HFD-induced obesity mice. In conclusion, our data suggest that HFD induces a classical reaction of microglia, characterized by an enhanced phosphorylation of p-38 and increased CD16/32 expression, which may in part contribute to increased nociceptive responses in HFD-induced obesity mice.