Biomaterials to be used as cell delivery systems for cardiac tissue engineering should be able to comply with cardiac muscle contractile activity, while favoring cell survival and neo-angiogenesis in a hostile environment. Biocompatible synthetic materials can be tailored to mimic cardiac tissue three-dimensional organization in the micro- and nanoscales. Nonetheless, they usually display mechanical properties that are far from those of the native myocardium and thus could affect host cell survival and activity. In the present investigation, inert poly-ε-caprolactone planar layers were manufactured to change the surface stiffness (with Young's modulus ranging from 1 to 133 MPa) without changing matrix chemistry. These substrates were challenged with neonatal murine cardiomyocytes to study the possible effect of substrate stiffness on such cell behavior without changing biological cues. Interestingly, softer substrates (0.91±0.08 and 1.53±0.16 MPa) were found to harbor mostly mature cardiomyocytes having assembled sarcomeres, as shown by the expression of alpha actinin and myosin heavy chain in typical striations and the upregulation of sarcomeric actin mRNA. On the other hand, a preferential expression of immature cardiac cell genes (Nkx-2.5) and proteins (GATA-4) in cardiac cells grown onto stiffer materials (49.67±2.56 and 133.23±8.67 MPa) was detected. This result could not be ascribed to significant differences in cell adhesion or proliferation induced by the substrates, but to the stabilization of cardiomyocyte differentiated phenotype induced by softer layers. In fact, cardiac cell electromechanical coupling was shown to be more organized on softer surfaces, as highlighted by connexin 43 distribution. Moreover, a differential regulation of genes involved in extracellular matrix remodeling was detected on soft films (0.91±0.08 MPa) as compared with the stiffest (133.23±8.67 MPa). Finally, the upregulation of a number of genes involved in inflammatory processes was detected when the stiffest polymer is used. These events highlight the differences in cell mechanosensitivity in a heterogeneous cell preparation and are likely to contribute to the differences encountered in cardiac cell phenotype induced by substrate stiffness.