Vascular smooth muscle cells (VSMCs) in the normal vascular wall regulate vascular contraction and dilation due to their contractility. But they change their phenotype from contractile to synthetic state and actively remodel the vascular wall in pathological conditions. Findings on the phenotypic change mechanism of VSMCs have been reported by many in vitro studies, however, mechanical environments in vivo vascular wall are quite different from those of in vitro culture condition: VSMCs in vivo exhibit an elongated shape and form a tissue that aligns with the circumferential direction of the walls, while VSMCs in vitro spread randomly and form irregular shapes during cultivation on flat culture dishes, and dedifferentiate into synthetic phenotype. To clarify the mechanisms of the phenotypic changes in VSMCs, it is essential to develop a cell culture model that consider the mechanical environment of in vivo vascular wall. Here, we fabricated the polydimethylsiloxane (PDMS)-based micro-grooved substrate with 5 or 20 μm of groove width and 5 μm of groove depth to induce cell elongation and alignment observed in vivo. We established the coating method with cell adhesion protein only on the surface of groove concaves, and found that VSMCs adhering into the concaves formed uniform cell tissue and allowed remarkable elongation. In particular, the micro grooves with 5 μm groove width and depth facilitated a significant nuclear deformation and volume reduction of the nucleus due to a lateral compression by the side wall of the groove concaves that is relatively similar to a sandwich-like arrangement of in vivo elastic lamellae, resulting in the cell proliferation inhibition and VSMC differentiation. These results indicate that our cell culture model with the micro-grooved substrates can be useful to study the mechanisms of the phenotypic changes in VSMCs under consideration of in vivo vascular mechanical environment.