Endothelial dysfunction is an early and central feature of atherosclerosis. Dietary resveratrol (RSV), a class of flavonoid compounds, have been demonstrated to exert several beneficial effects on human body. In this study, we investigated the protective effects of RSV on high fat diet-induced endothelial dysfunction. Human aortic endothelial cells (HAECs) were treated with RSV to evaluate the gene expression of the endothelial nitric oxide synthase (eNOS). Apolipoprotein E (apoE-/-) mice were fed a high-fat, high-cholesterol diet (HCD) or HCD supplemented with RSV for 8 weeks. Treatment of cultured HAECs with RSV dose-dependently upregulated the eNOS expression as assessed by quantitative RT-PCR and Western blot, respectively. In addition, RSV increased the promoter activity of the human eNOS gene, as determined by luciferase assays of the eNOS promoter gene. The cAMP-response element binding protein (CREB) was identified as the target transcription factor involved in the RSV mediated upregulation of eNOS expression. RSV increased phosphorylation of CREB through protein kinase A (PKA) activation, which induced a CREB-mediated upregulation of eNOS transcription. Consequently, RSV treatment significantly reversed the deleterious effects of oxidized LDL (oxLDL)-induced oxidative stress in HAECs. In vivo, treatment with RSV improves endothelial dysfunction and attenuates atherosclerotic plaque formation in apoE-/- mice through PKA-CREB-dependent pathway. Our findings demonstrate that RSV has an effect of activating eNOS expression, contributing to the prevention of dyslipidemia-induced endothelial dysfunction and atherosclerosis.