Abstract Background Retinoic acid-related orphan receptor α (RORα) has been implicated in the progression of atherosclerosis, but its role in the proliferation of vascular smooth muscle cells (vSMCs) has not been fully examined. We previously reported that RORα activates AMP-activated protein kinase (AMPK), which is associated with the suppression of vSMC proliferation. Therefore, we investigated the suppressive function of RORα on the proliferation of vSMCs and the molecular mechanisms involved. Results First, RORα and its activator, cholesterol sulfate (CS), induced the activation of AMPK in both human aortic SMCs and rat A7r5 cells, which was accompanied by the suppression of mammalian target of rapamycin (mTOR) and p70 ribosomal protein S6 kinase 1. Second, RORα and CS modulated the expression of cell-cycle-regulating factors, such as p53, p27, and cyclin D in vSMCs. Consistent with this, the overexpression of RORα or CS treatment suppressed the proliferation of human aortic SMCs and rat A7r5 cells, possibly through G1 arrest. RORα and CS also inhibited the migration of A7r5 cells in two-dimensional and three-dimensional cell migration assays. Finally, we demonstrated that the infusion of adenovirus encoding RORα into arteries suppressed neointima formation after balloon injury in rats. Conclusion These results demonstrate that RORα inhibits vSMC proliferation through AMPK-induced mTOR suppression, and suggest that RORα is a therapeutic target for the cardiovascular diseases associated with vSMC proliferation.