Visceral hypersensitivity is one of the most prevalent symptoms of inflammatory bowel disease (IBD), and it can be difficult to cure despite achieving endoscopic remission. Accumulating studies have described that macrophage colony-stimulating factor (M-CSF) modulates neuroinflammation in the central nervous system (CNS) and the development of chronic pain, while the underlying mechanism for whether and how M-CSF/CSF1R signaling pathway regulates visceral hypersensitivity following colitis remains unknown. In the present study, using the dextran sulfate sodium (DSS)-induced colitis model, we determined that microglial accumulation occurred in the spinal dorsal horn during remission phase. The reactive microglia released inflammatory factor, increased neuronal excitability in the dorsal horn, and produced chronic visceral pain behaviors in DSS-treated adult male mice. In addition, we also found significantly increased signaling mediated by astrocytic M-CSF and microglial CSF1R in dorsal horn in the mice with colitis. Exogenous M-CSF induced microglial activation, neuronal hyperactivity and behavioral hypersensitivity in the control group, inhibition of astrocyte/microglia by fluorocitrate/minocycline significantly suppressed microglial and neuronal activity, and relieved the visceral hypersensitivity in the model mice. Overall, our experimental study uncovers the critical involvement of spinal astrocyte-derived M-CSF and reactive microglia in the initiation and maintenance of visceral hypersensitivity following colitis, thereby identifying spinal M-CSF as a target for treating chronic visceral pain. This may provide more accurate theoretical guidance for clinical patients with IBD.