The function of microRNAs (miRNAs or miRs) in regulating neuropathic pain has attracted increasing attention in recent years. However, the precise mechanism of miRNAs in neuropathic pain remains largely unknown. In the present study, an important role of miR-141 and its putative target gene, high-mobility group box-1 (HMGB1), was demonstrated in a rat model of neuropathic pain induced by chronic constriction injury (CCI). The expression of miR-141 was significantly downregulated in the dorsal root ganglion of rats following CCI surgery. Overexpression of miR-141 by intrathecal injection of miR-141 precursor mediated by a lentivirus-derived gene transfer significantly inhibited mechanical allodynia, thermal hyperalgesia and proinflammatory cytokine release in CCI rats. Using a dual luciferase reporter assay, a direct interaction between miR-141 and the 3'-untranslated region of HMGB1 was verified. Overexpression of miR-141 significantly suppressed the expression of HMGB1 in vitro and in vivo. Furthermore, overexpression of HMGB1 apparently abrogated the beneficial effect of miR-141 on inhibiting neuropathic pain. Taken together, the data suggest that overexpression of miR-141 alleviates neuropathic pain development via targeting and inhibiting HMGB1, implying that blocking HMGB1 by miR-141 could be a useful therapeutic strategy for the treatment of neuropathic pain.