Cholesterol and chronic activation of hypoxia-inducible factor-1 (HIF-1) have been separately implicated in the pathogenesis and progression of liver diseases. In AML12 hepatocytes increased HIF-1α protein accumulation was evident after 2 h of incubation with cholesterol, whereas enhanced HIF-1 transcriptional activity was observed after 6 h. Investigations into the molecular mechanism have shown that cholesterol inhibited HIF-1α degradation. Mitochondrial dysfunction and enhanced mitochondrial reactive oxygen species (ROS) generation were observed in 2-h cholesterol-treated cells along with augmented nitric oxide (NO) levels. Further analysis indicated that HIF-1α stabilization at later time (6 h), but not after 2 h, of incubation with cholesterol was dependent on NO production. To elucidate the role of mitochondrial dysfunction in HIF-1α stabilization, mitochondrial DNA-depleted hepatocytes were prepared. In these cells the ability of cholesterol to activate the HIF-1 pathway was abolished. Similarly, catalase overexpression also attenuated cholesterol-induced HIF-1α accumulation. These results demonstrate that cholesterol promotes HIF-1 activation in a ROS- and NO-dependent manner. Chronic liver activation of HIF-1 by cholesterol may mediate its deleterious effects in the liver.