High-mobility group box 1 (HMGB1) protein is a multifunctional cytokine involved in inflammatory responses and tissue repair. In this study, it was examined whether HMGB1 plays a role in skin wound repair both in normoglycemic and diabetic mice. HMGB1 was detected in the nucleus of skin cells, and accumulated in the cytoplasm of epidermal cells in the wounded skin. Diabetic human and mouse skin showed more reduced HMGB1 levels than their normoglycemic counterparts. Topical application of HMGB1 to the wounds of diabetic mice enhanced arteriole density, granulation tissue deposition, and accelerated wound healing. In contrast, HMGB1 had no effect in normoglycemic mouse skin wounds, where endogenous HMGB1 levels may be adequate for optimal wound closure. Accordingly, inhibition of endogenous HMGB1 impaired wound healing in normal mice but had no effect in diabetic mice. Finally, HMGB1 had a chemotactic effect on skin fibroblasts and keratinoyctes in vitro. In conclusion, lower HMGB1 levels in diabetic skin may play an important role in impaired wound healing and this defect may be overcome by the topical application of HMGB1.