The commercial use of epidermal growth factor (EGF) is extensive and has been shown to be effective for skin wound healing in clinical practice. There is evidence to indicate that the topical administration of EGF significantly accelerates re-epithelialization by promoting keratinocyte mitogenesis and migration following acute injury; however, the mechanisms involved remain to be elucidated. Thus, in this study, we focused on Kindlin-1, a four-point-one, ezrin, radixin, moesin (FERM)-domain-containing adaptor protein, and report its contribution to EGF-induced re-epithelialization in skin wound healing. In tissue samples, the expression of Kindlin-1 was induced upon EGF treatment compared to that in the natural healing group. In immortalized human keratinocytes (HaCaT cells), we further proved that Kindlin-1 was necessary for mediating EGF-induced activation signals, including integrin β1 activation, focal adhesion kinase (FAK) phosphorylation and actin re-organization, which finally led to enhanced cell proliferation and migration. These results indicate that Kindlin-1 is essential in EGF-induced re-epithelialization in skin wound healing and provide additional rationale for the clinical application of EGF in the treatment of acute wounds.