Aim To explore the protective effects of extract of leaves of Ginkgo biloba (EGb) on vascular dysfunction injured by high lipidemia in rats and analyze the possible mechanism related to paraoxonase (PON-1) activity. Methods 32 Male Sprague-Dawley rats were divided randomly into four groups. The rats of control group and high lipid (HL) diet group were fed with conventional chow or fat emulsion (contained 10% cholesterol and 20% lard) respectively. The other two groups were fed with fat emulsion plus EGb [0.25 mg/(kg·d)] and vitamin E [100 mg/(kg·d)] respectively. In order to potentiate atherogenic effects of fat emulsion,the rats were received an extra vitamin D3 (thirty hundred thousand U/kg, orally, for 2 days) before feeding with fat emulsion. Animal was killed in the 21st day and aortic artery was isolated to analyze response of vascular function. Fasting blood samples were collected after experiment for an assay of lipids,nitri oxide (NO),malondialdehyde (MDA) and PON-1 activity. Results The HL diet resulted in a hyperlipidemia (HLA) characterized by increase of total cholesterol (TC), triglyceride (TG), LDLC, and atherosclerosis index (AI) [AI=(TC-HDLC)/HDLC]. The treatment of both of EGb and vitamin E have no significant effect on HL diet induced-HLA. The HLA significantly inhibited endothelium-dependent relaxation (EDR) (maximum relaxation: 95.1%±19.8% vs 47.1%±14.9% respectively in control and HL diet group,p<0.01) and endothelium-independent relaxation (maximum relaxation: 98.2%±3.6% vs 56.7%±7.9% respectively in control and HL diet group,p<0.01). Both EGb and vitamin E prevented from decrease of EDR (81.8%±9.3%, 76.2%±11.3% respectively in EGb and vitamin E group,p<0.01 vs HL diet group,p>0.05 vs control group) and endothelium-independent relaxation (85.8%±7.2%, 83.6%±4.9% respectively in EGb and vitamin E group,p<0.01 vs HL diet group,p>0.05 vs control group ). The HLA markedly decreased the concentration of both NO (3.35±1.07 μmol/L vs 5.16±1.32 μmol/L respectively in HL diet and control group,p<0.01) and PON-1 activity (kU/L ) (7.9±3.74 vs 18.03±7.24 respectively in HL diet and control group,p<0.01) and increased plasma MDA (4.37±1.29 μmol/L vs 2.61±0.94 μmol/L respectively in HL diet and control group, p<0.01) . EGb and vitamin E preserved PON-1 activity (16.5±5.25 and 14.6±3.83 respectively in EGb and vitamin E group, p<0.01 or p<0.05 vs HL diet group, p>0.05 vs control group) and plasma NO (5.05±1.41 and 4.91±1.65 respectively in EGb and vitamin E, p<0.01 vs HL diet group, p>0.05 vs control) and significantly blocked elevation of MDA (2.77±1.30 and 2.88±1.17 respectively in EGb and vitamin E, p<0.05 vs HL diet group, p>0.05 vs control group) induced by HLA. Conclusion EGb exerted protective effects against HLA-impaired both EDR and endothelium-independent relaxation. The mechanisms of protection of EGb against vascular dysfunction are related to preserving activity of PON-1 and reducing production of lipid peroxidation.