Endothelial cell (EC) dysfunction contributes to atherosclerosis, which is associated with arterial stiffening and fibronectin (FN) deposition, by ECs and smooth muscle cells (SMCs). The effect of stiffness on the EC/FN interaction and fibrillar adhesion formation has been poorly studied. An in vitro model was prepared that included FN-coated polydimethylsiloxane (PDMS) films with similar hydrophobicity and roughness but distinct Young's modulus values, mimicking healthy (1.0 MPa) and atherosclerotic (2.8 MPa) arteries. Human aortic abdominal endothelial cells (HAAECs) seeded on 1.0 MPa PDMS films spread over time and reached their maximum surface area faster than on 2.8 MPa PDMS films. In addition, HAAECs appeared to organize focal adhesion more rapidly on 1.0 MPa PDMS films, despite the similar cell binding domain accessibility to adsorbed FN. Interestingly, we also observed up to a ~5-fold increase in the percentage of HAAECs that had a well-developed fibrillar adhesion on 1.0 MPa compared to 2.8 MPa PDMS films as verified by integrin α5 subunits, tensin, and FN staining. This variation did not affect EC migration. These results suggest that there are favourable conditions for FN matrix assembly by ECs in early atherosclerosis rather than at advanced stages. Our in vitro model will therefore be helpful to understand the influence of bulk stiffness on cells involved in atherosclerosis.