Intrathecal treatment with recombinant high-mobility group box-1 (rHMGB1) in naive mice leads to a persistent and significantly decreased hind paw withdrawal threshold to mechanical stimuli, suggesting that spinal HMGB1 evokes abnormal pain processing. By contrast, repeated intrathecal treatment with anti-HMGB1 antibody significantly reverses hind paw mechano-hypersensitivity in mice with a partial sciatic nerve ligation (PSNL). By contrast, the cellular mechanism by which spinal HMGB1 induces neuropathic pain has yet to be fully elaborated. The current study tested the hypothesis that spinal HMGB1 could induce mechanical hypersensitivity through the activation of specific receptor in glial cells. Intrathecal pretreatment with toll-like receptor (TLR) 4 inhibitors, but not TLR5, receptor for advanced glycation end-products and C-X-C chemokine receptor type 4 inhibitors, prevented rHMGB1-evoked mechanical hypersensitivity. Activation of spinal astrocytes appears to be crucial for the mechanism of action of rHMGB1 in naive mice, as intrathecal pretreatment with astrocytic inhibitors prevented the rHMGB1-induced mechanical hypersensitivity. Interleukin-1 beta (IL-1 beta) was up-regula