Chemotherapy-induced neuropathic pain (CNP) is the major dose-limiting factor in cancer chemotherapy. However, the mechanisms underlying CNP remain elusive. In the present study, CNP was induced by repeated intraperitoneal injection of vincristine (VCR) into male C57BL/6J mice. VCR administration caused significant activation of Wnt/beta-catenin signaling, which led to the activation of astrocytes, microglia, the release of inflammatory cytokines tumour necrosis factor (TNF)-alpha, monocyte chemoattractant protein-1 (MCP-1) and the activation of subsequent mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) signaling pathway in CNP mice. Blocking Wnt/beta-catenin signaling by intrathecal administration of the inhibitors of Wnt response (IWR) effectively attenuated VCR-induced neuropathic pain. Furthermore, IWR inhibited the activation of astrocytes, microglia, TNF-alpha, MCP-1 and MAPK/ERK signaling in the spinal cord, which was triggered by VCR-induced Wnt/beta-catenin signaling upregulation. These results suggest that Wnt/beta-catenin signaling plays a critical role in VCR-induced neuropathic pain and provides evidence for potential interfering with Wnt/beta-catenin signaling to ameliorate VCR-induced neuropathic pain.