Endothelial dysfunction is a key early step in atherosclerosis. 25-hydroxycholesterol (25-OHC) is found in atherosclerotic lesions. However, whether 25-OHC promotes atherosclerosis is unclear. Here we hypothesize that 25-OHC, a proinflammatory lipid, can impair endothelial function, which may play an important role in atherosclerosis. Bovine aortic endothelial cells (BAECs) were incubated with 25-OHC. Endothelial cell proliferation, migration and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation were determined. The expression and phosphorylation of endothelial nitric oxide synthase (eNOS) and AKT as well as the association of eNOS and heat shock protein 90 (HSP90) were detected by immunoblotting and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining, the caspase 3 activity and the expression of Bcl-2, Bax, the cleaved caspase 9 and cleaved caspase 3 were detected by immunoblotting. Finally, aortic ring from SD rats were isolated and treated with 25-OHC and the endothelium-dependent vasodilation was evaluated. 25-OHC significantly inhibited endothelial cell proliferation, migration and tube formation. 25-OHC markedly decreased NO production and increased superoxide anion generation. 25-OHC reduced the phosphorylation of AKT and eNOS and the association of eNOS and HSP90. 25-OHC also enhanced endothelial cells apoptosis by decreasing Bcl-2 expression and increasing cleaved caspase 9 and cleaved caspase 3 expressions and the caspase 3 activity. 25-OHC impaired endothelium-dependent vasodilation. These data demonstrated that 25-OHC could impair endothelial function by uncoupling and inhibiting eNOS activity as well as by inducing endothelial cell apoptosis. Our findings indicate that 25-OHC may play an important role in regulating atherosclerosis.