Wound healing is a basic biological process restoring the integrity of the skin. The role of microRNAs (miRNAs) during this process remains largely unexplored. By using an in vivo human skin wound healing model, we show here that the expression of miR-31 is gradually up-regulated in wound edge keratinocytes in the inflammatory (1 day after injury) through the proliferative phase (7 days after injury) in comparison with intact skin. In human primary keratinocytes, overexpression of miR-31 promoted cell proliferation and migration, while inhibition of miR-31 had the opposite effects. Moreover, we identified epithelial membrane protein 1 (EMP-1) as a direct target of miR-31 in keratinocytes. The expression of EMP-1 in the skin was negatively correlated with the level of miR-31 during wound healing. Silencing of EMP-1mimicked the effects of overexpression of miR-31 on keratinocyte proliferation and migration, indicating that EMP-1 is a critical target mediating the functions of miR-31 in keratinocytes. Finally, we demonstrated that TGF-β2, which is highly expressed in skin wounds, up-regulated miR-31 expression in keratinocytes. Collectively, we identify miR-31 as a key regulator for promoting keratinocyte proliferation and migration during wound healing.Journal of Investigative Dermatology accepted article preview online, 16 February 2015. doi:10.1038/jid.2015.48.