We have shown that autocrine proliferation of human keratinocytes (KCs) is strongly dependent upon amphiregulin (AREG), whereas blockade of heparin-binding EGF-like growth factor (HB-EGF) inhibits KC migration in scratch wound assays. Here we demonstrate that expression of soluble HB-EGF (sHB-EGF) or full-length transmembrane HB-EGF (proHB-EGF), but not proAREG, results in profound increases in KC migration and invasiveness in monolayer culture. Coincident with these changes, HB-EGF significantly decreases mRNA expression of several epithelial markers including keratins 1, 5, 10, and 14 while increasing expression of markers of cellular motility including SNAI1, ZEB1, COX-2, and MMP1. Immunostaining revealed HB-EGF-induced expression of the mesenchymal protein vimentin and decreased expression of E-cadherin, as well as nuclear translocation of β-catenin. Suggestive of a trade-off between KC motility and proliferation, overexpression of HB-EGF also reduced KC growth by >90%. We also show that HB-EGF is strongly induced in regenerating epidermis after partial-thickness wounding of human skin. Taken together, our data suggest that expression of HB-EGF in human KCs triggers a migratory and invasive phenotype with many features of epithelial–mesenchymal transition (EMT), which may be beneficial in the context of cutaneous wound healing.