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This study was carried out to investigate that the effect of grape seed oil (GSO) on Hypercholesterolemia in rats. Thirty-five rats were divided into 5 groups, group 1 was given the basal diet as a negative control group (-ve), group 2 was given the basal diet with GSO instead of soybean oil as a negative control for GSO. Groups (3, 4 and 5) were fed on high cholesterol diet (HCD) (1% cholesterol powder and 0.5% bile salt for 8 weeks), Group 3 was +ve control, group 4 and 5 were treatment groups which received GSO 2% and 4% daily with HCD. The results of this study indicated that the GSO caused an improvement in the blood lipids especially 4% GSO. The 4% GSO decreased serum TC, TG, LDL-c and VLDL significantly (P ˂ 0.05), and caused a significant increase in HDL-C level. Also, 2% GSO decreased TG, LDL-C and VLDL significantly (P ˂ 0.05), whereas the level of HDL-C showed significant increases. Subsequently, GSO enhanced the lipid ratios: atherogenic coefficient (AC), cardiac risk ratio (CRR), LDL-c to HDL-c ratio and atherogenic index of plasma (AIP). Moreover, the serum, liver function (AST, ALT and ALP) levels also, enhanced in the GSO groups. However, 4% GSO led to significant decrease in serum MDA and elevated serum GST. Otherwise, histopathological examination showed enhanced in the heart and aorta of rats compared with the +ve control group. This study indicates that GSO effective in lowering total cholesterol, triglyceride and LDL-c and increasing HDL-c. Therefore, GSO have hypocholesterolemic effect and might be effective to protect against the risk of CVD.