It has been shown that epigenetic regulation plays an important role in skin wound healing. We previously found that histone H3K27me3 demethylase JMJD3 regulates inflammation and cell migration in keratinocyte wound healing. In this study, we identified Notch1 as a direct target of JMJD3 and NF-κB in wounded keratinocytes using in vitro cell and in vivo animal models. We found that Notch1 is up-regulated in the wound edge and its expression is dependent on JMJD3 and NF-κB in wounded keratinocytes. We also found that Notch1 activates the expression of RhoU and PLAU gene, which are critical regulators of cell migration. Consistently, depletion or inactivation of Notch1 resulted in decreased filopodia formation, increased focal adhesion and actin stress fiber, leading to reduced keratinocyte migration and skin wound healing. Thus, our findings provide the molecular mechanism involving JMJD3/NF-κB-Notch pathway in keratinocyte wound healing.