Human epithelial cells are permanently stimulated by external mechanical forces. The present in vitro study suggests that keratinocytes respond to mechanical strain by a coordinated spatial and functional utilization of β1-integrins and the epidermal growth factor receptor (EGFR) with impact to the adhesion properties. It was found that a single mechanical stretch applied to HaCaT keratinocytes elevates the substrate adhesion, in particular to fibronectin and collagen type IV but not to laminin indicating the relevance of β1-integrins in this process. This was confirmed using a functional blocking antibody directed against β1-integrins which reversed the stretch-induced adhesion. Furthermore, mechanical stretch gives rise to a rapid redistribution of β1-integrins in clusters on the basal cell membrane, without changing the overall amount of this particular integrin subset. Concomitantly, the EGFR co-localizes with β1-integrin suggesting a functional cooperation of both membrane proteins in mechano-signaling. This is corroborated by data showing that stretch-induced activation of the EGFR and the downstream element extracellular regulated kinase 1/2 (ERK1/2) is reversed by preincubation with β1-integrin antibodies. Vice versa, blocking the EGFR using a specific inhibitor abrogates stretch-induced ERK1/2 activation. In summary, these results show a functional cooperation of β1-integrins and EGFR in the adhesion complex supporting the transmission of stretch-induced signals.