The principal objective of this experiment was to evaluate the anti-atherosclerotic activity of hempseed water extract in apolipoprotein E knockout (ApoE KO) mice. Fourteen male ApoE KO mice were divided into 2 groups and were treated via intragastric inoculation for 14 weeks. The hempseed water extract (HWE) inoculation group exhibited greater gains in weight as compared to the control group, which was inoculated with distilled water. Plaque lesion areas in the aortic sinus were reduced in the HWE group. Total plasma cholesterol, LDL-cholesterol, atherosclerotic index, and cardiac risk factor were all reduced in the HWE group. These results demonstrate that HWE is an excellent nutritional resource and evidences anti-atherosclerotic activity in ApoE KO mice. Further studies will be required to assess the pharmacological mechanisms underlying these effects.