Object: Atherosclerotic vascular disease is the leading cause of mortality in the industrialized world. Even after angioplasty and stent placement, neointimal hyperplasia developed from phenotypic modulation toward a proliferative and dedifferentiated, leads to pathogenic vessel lumen constriction and restenosis. Melatonin was first known to regulate the circadian and seasonal rhythm. Mounting evidences have revealed that melatonin functions as an antioxidant or immune enhancer, however, there has not yet been much research into the effect of melatonin on vascular smooth muscle cell growth Methods: Rat aortic smooth muscle cells were isolated from a 4-week-old Sprague-Dawley male rat and cultured in low-glucose DMEM supplemented with 20% fetal bovine serum (FBS). Primary VSMCs were seeded in a Seahorse XF24 plate, serum starved for 24 h, and incubated with or without FBS or melatonin (2mM) for 24 h. Then, the basal ECAR and OCR were measured and automatically calculated using a Seahorse XF24 analyzer. Results: Activation of VSMCs with FBS increased their proliferation, which was prevented by melatonin treatment. In addition, the VSMCs were impeded at the G1 phase in the melatonin-treated group. We found that melatonin caused significant decrease in the major parameters of mitochondrial function including a decrease in basal OCR and maximal respiration, as well as ATP-linked respiration, and the subsequent induction of Sestrin2. Finally, melatonin -induced Sestrin2 upregulation inhibited proliferation, and also apoptosis via limiting excessive ROS generation. Conclusion: Taken together, we demonstrate that FBS-stimulated VSMCs plays an important role in their proliferation and suggest that melatonin can be a potential therapeutic target to prevent vessel lumen constriction during the course of atherosclerosis and restenosis.