Vascular smooth muscle cell (VSMC) apoptosis plays an essential role in vascular development and atherosclerosis. Hyperglycemia inhibits VSMC apoptosis, which may contribute to the development of diabetic vasculopathy. In the present study, we analyzed the mechanism of high-glucose-induced anti-apoptotic effect in cultured human aortic smooth muscle cells (HASMCs). Compared with normoglycemia, exposure of HASMCs to hyperglycemia but not mannitol significantly increased sphingosine kinase 1 (SK1) activity but not SK2 activity. This increase was inhibited by protein kinase C (PKC) inhibitor GF109203X, the antioxidant N-acetylcysteine, and the reduced form of glutathione. The mechanism of SK1 activation by high glucose involves plasma membrane translocation. In addition, hyperglycemia markedly inhibited serum withdrawal-induced apoptosis in HASMCs. Importantly, inhibition of SK1 by either a competitive inhibitor N',N'-dimethylsphingosine or expression of dominant-negative mutant of SK1(G82D) or specific small interference RNA knockdown substantially attenuated hyperglycemia-induced anti-apoptotic effect and anti-apoptotic protein Bcl-2 expression in HASMCs. Moreover, SK1-mediated anti-apoptotic effect requires the intracellular effects of sphingosine-1-phosphate. We conclude that hyperglycemia stimulates SK1 activity via PKC- and oxidative stress-dependent pathways, leading to decreased apoptosis in HASMCs. Taken together, these observations have important implications for understanding the roles of the SK1 signaling pathway in the pathogenesis of diabetic vasculopathy.