Chronic treatment with vincristine (VCR) causes mechanical allodynia as an adverse effect. We previously reported that peripheral macrophage-derived interleukin-6 played a critical role in VCR-induced allodynia. However, the involvement of glial cell activation and central sensitization in VCR-induced allodynia is still unclear. In this study, we focused on tumor necrosis factor-alpha (TNF-α) in spinal cord, and investigated the role of TNF-α in VCR-induced allodynia in mice. VCR (0.1mg/kg, i.p.) was administered to mice once per day for 7 days. The expression of TNF-α mRNA and the protein in spinal cord was evaluated by quantitative real-time PCR and immunohistochemistry, respectively. In VCR-treated mice, TNF-α mRNA gradually increased and was significantly up-regulated on day 7. As measured by immunohistochemistry, microglia and astrocytes were activated in the spinal dorsal horn on day 7 of VCR administration. The immunoreactivity of TNF-α was co-localized in some of the activated microglia and astrocytes. In behavioral analysis, a neutralizing antibody of TNF-α, which was injected intrathecally on days 0, 3, and 6, significantly attenuated VCR-induced mechanical allodynia on days 4 and 7. These results suggest that VCR treatments elicited the activation of glial cells in spinal cord, and up-regulated TNF-α in these cells may play an important role in VCR-induced mechanical allodynia.