Keratinocyte growth factor (KGF) and α5β1-integrin are not expressed in normal skin but they are both highly upregulated in the migrating epidermis during wound healing. Here we report that KGF increased α5 mRNA and protein levels in epidermoid carcinoma cells and stratified bioengineered epidermis. Interestingly, KGF increased integrin α5 in the basal as well as suprabasal cell epidermal layers. Promoter studies indicated that KGF-induced integrin α5 promoter activation was dependent on the C/EBP transcription factor binding site. Accordingly, KGF induced sustained phosphorylation of C/EBP-β that was dependent on activation of ERK1/2. In addition, a dominant negative form of C/EBP-β inhibited α5 promoter activity and blocking C/EBP-β with siRNA diminished integrin α5 expression. Taken together, our data indicate that KGF increased integrin α5 expression by phosphorylating C/EBP-β. Interestingly, KGF-induced upregulation of integrin α5 was more pronounced in three-dimensional tissue analogues than in conventional two-dimensional culture suggesting that stratified epidermis may be useful in understanding the effects of growth factors in the local tissue microenvironment.