Fibroblast differentiation is an essential step during wound healing and fibrosis. Fibronectin (FN) is a major component of the extracellular matrix and occurs in two main forms: plasma and cellular FN. The latter includes the alternatively spliced domain A (EDA). Although EDA-containing cellular fibronectin (EDA-FN) is associated with fibroblast differentiation, how EDA-FN promotes differentiation is incompletely understood. In this study, we investigate the mechanism by which EDA-FN contributes to fibroblast differentiation with emphasis on the characterization of the EDA-FN receptor. We show that EDA-FN increases α-SMA expression (immunofluorescence), collagen deposition, cell contractility, and focal adhesion kinase (FAK) activation (immunoblotting); whereas plasma FN, a form lacking EDA, shows no effect. Primary lung fibroblasts constitutively express α 4 β 7 integrin receptor (FACS and RT-PCR). Blocking of α 4 β 7 reduces fibroblast adhesion to EDA-FN and inhibits α-SMA expression, collagen deposition, and FAK activation induced by EDA-FN. Using recombinant EDA-containing peptides, we demonstrate that the EDA segment is sufficient to induce fibroblast differentiation via binding to α 4 β 7. EDA-FN induces MAPK-Erk1/2 activation and inhibition of MEK1/2 attenuates EDA-FN-induced α-SMA expression. Our findings demonstrate that EDA-FN induces fibroblast differentiation by a mechanism that involves binding of EDA to α 4 β 7 integrin followed by activation of FAK and MAPK-associated signaling pathways.—Kohan, M., Muro, AF, White, ES, Berkman, N. EDA-containing cellular fibronectin induces fibroblast differentiation through binding to α 4 β 7 integrin receptor and MAPK/Erk 1/2-dependent signaling.