Endothelial-to-mesenchymal transition (EndMT), during which endothelial cells (ECs) transdifferentiate into mesenchymal phenotype, plays a key role in the development of vascular implant complications such as endothelium dysfunction and in-stent restenosis. Substrate stiffness has been confirmed as a key factor to influence EC behaviors; however, so far, the relationship between substrate stiffness and EndMT has been rarely studied. Here, ECs were cultured on the (poly(L-lysine)/hyaluronate acid) (PLL/HA) multilayer films with controlled stiffness for 2 weeks, and their EndMT behaviors were studied. We demonstrated that ECs lost their markers (vWf and CD31) in a stiffness-dependent manner even without supplement of growth factors, and the softer film favored the maintaining of EC phenotype. Further, induced by transforming growth factor beta 1 (TGF-beta 1), ECs underwent EndMT, as characterized by losing their typical cobblestone morphology and markers and gaining smooth muscle cell markers (alpha-smooth muscle actin and calponin). Interestingly, stronger EndMT was observed when ECs were cultured on the stiffer film. Collectively, our findings suggest that substrate stiffness has significant effects on EndMT, and a softer substrate is beneficial to ECs by keeping their phenotype and inhibiting EndMT, which presents a new strategy for surface design of vascular implant materials.