Abnormal VSMCs proliferation is common pathological aspect in various vascular diseases such as atherosclerosis, hypertension, and restenosis. According to previous studies, inhibition of VSMC proliferation plays a central role in prevention of vascular diseases. It has been established that AMPK, a key regulator of cellular homeostasis, regulates VSMCs migration and proliferation. In recent studies, TGF-β1 has proliferative capacity in VSMCs through Smad2/3 phosphorylation. Chloroquine (CQ) and hydroxychloroquine (HCQ), anti-malarial drugs, have an effect on cell proliferation in cardiovascular disease and cancer. Nevertheless, the effect of CQ and HCQ to VSMCs proliferation and its target has not revealed yet. Therefore, this study aims to investigate the effects of anti-malarial drugs, CQ and HCQ, on inhibition of VSMCs proliferation via AMPK and neointimal proliferation in mice. In VSMCs, CQ and HCQ increased AMPK phosphorylation in a dose-dependent manner. Also, CQ and HCQ suppressed PDGF-induced VSMCs proliferation and cell cycle progression. In addition, CQ and HCQ inhibited Smad3 phosphorylation and VSMC proliferation induced by TGF-β1 treatment. To confirm the effect of CQ and HCQ in vivo, mice carotid arteries were ligated and treated with CQ or HCQ every other day for 3 weeks. CQ and HCQ have been shown to suppress neointimal proliferation in a mouse model of carotid artery ligation-induced neointima formation. In conclusion, CQ and HCQ inhibited cell proliferation and cell cycle progression in VSMCs through AMPK activation and Smad3 inhibition. Moreover, intima thickness induced by carotid artery ligation was reduced in mouse arteries treated with CQ and HCQ compared to vehicle control. These results suggest that CQ and HCQ might be a potential therapeutic agent against vascular diseases with abnormal VSMCs proliferation.