Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a common feature of disease progression in atherosclerosis. Cell proliferation is regulated by cell cycle regulatory proteins. MicroRNAs (miR) have been reported to act as important gene regulators and play essential roles in the proliferation and migration of VSMCs in a cardiovascular disease. However, the roles and mechanisms of miRs in VSMCs and neointimal formation are far from being fully understood. In this study, cell cycle-specific cyclin D1 was found to be a potential target of miR-365 by direct binding. Through an in vitro experiment, we showed that exogenous miR-365 overexpression reduced VSMC proliferation and proliferating cell nuclear antigen (PCNA) expression, while miR-365 was observed to block G1/S transition in platelet-derived growth factor-bb (PDGF-bb)-induced VSMCs. In addition, the proliferation of VSMCs by various stimuli, including PDGF-bb, angiotensin II (Ang II), and serum, led to the downregulation of miR-365 expression levels. The expression of miR-365 was confirmed in balloon-injured carotid arteries. Taken together, our results suggest an anti-proliferative role for miR-365 in VSMC proliferation, at least partly via modulating the expression of cyclin D1. Therefore, miR-365 may influence neointimal formation in atherosclerosis patients. J. Cell. Biochem. 115: 1752–1761, 2014. © 2014 Wiley Periodicals, Inc.