Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are important pathogenic mechanisms in atherosclerosis and restenosis after vascular injury. In this study, we investigated the effects of beta-lapachone (beta L) (3,4-Dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), which is a potent antitumor agent that stimulates NAD(P)H:quinone oxidoreductase (NQO)1 activity, on neointimal formation in animals given vascular injury and on the proliferation of VSMCs cultured in vitro. beta L significantly reduced the neointimal formation induced by balloon injury. beta L also dose-dependently inhibited the FCS- or platelet-derived growth factor-induced proliferation of VSMCs by inhibiting G(1)/S phase transition. beta L increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase 1 in rat and human VSMCs. Chemical inhibitors of AMPK or dominant-negative AMPK blocked the beta L-induced suppression of cell proliferation and the G(1) cell cycle arrest, in vitro and in vivo. The activation of AMPK in VSMCs by beta L is mediated by LKB1 in the presence of NQO1. Taken together, these results show that beta L inhibits VSMCs proliferation via the NQO1 and LKB1-dependent activation of AMPK. These observations provide the molecular basis that pharmacological stimulation of NQO1 activity is a new therapy for the treatment of vascular restenosis and/or atherosclerosis which are caused by proliferation of VSMCs. (Circ Res. 2009;104:842-850.)