We previously showed that wound-induced hypoxia is related to keratinocyte migration. The ability of keratinocytes within wound healing to undergo epithelial to mesenchymal transition (EMT) contributes significantly to the acquisition of migratory properties. However, the effect of hypoxia on keratinocyte EMT on wound healing and the potential mechanism are poorly documented. This study first demonstrated that reactive oxygen species (ROS) appear to be an essential signalling mediator in keratinocytes with increased EMT and migration subjected to hypoxic conditions. Next, we showed that the expression of sex-determining region Y-box 2 (SOX2), a stemness-associated molecule, is ROS-dependent under hypoxia and that SOX2 inhibition in keratinocytes dramatically prevented hypoxia-induced EMT and migration. In addition, β-catenin was found to be a potential molecular target of SOX2, and the activation of Wnt/β-catenin was required for hypoxia-induced EMT and migration. Using an in vitro skin culture model and an in vivo skin wound model, our study further reinforced the critical role of ROS in inducing EMT through SOX2 expression and subsequent activation of Wnt/β-catenin, allowing for rapid reepithelialization of the wound area. Taken together, our findings reveal a previously unknown mechanism by which hypoxia promotes wound healing by promoting reepithelialization through the production of ROS, inducing keratinocyte EMT and migration via the enhancement of SOX2 and activation of Wnt/β-catenin.Copyright © 2022 Yan Shi et al.