Apolipoprotein E (apoE) is a multifunctional protein synthesized by the liver and by tissue macrophages. Plasma apoE (derived primarily from the liver) regulates plasma lipoprotein metabolism, but macrophage-derived apoE was shown to slow the progression of atherosclerosis independent of plasma lipid levels. We utilized liver-directed gene transfer to test the hypothesis that hepatic expression of human apoE would inhibit atherogenesis even in a model in which apoE expression has little effect on plasma lipoproteins. LDL receptor-deficient mice fed a western-type diet for 5 weeks were injected with a second-generation recombinant adenovirus encoding human apoE3 or control virus. Plasma cholesterol levels were not significantly different in the two groups of mice after virus injection. Four weeks after injection, atherosclerosis was examined using three independent assays. Expression of apoE was associated with significantly reduced atherosclerosis compared with control mice in both the aortic arch (decreased by 43%) and the aortic root (decreased by 59%). In summary, hepatic overexpression of apoE inhibited progression of atherosclerosis in LDL receptor-deficient mice without reducing plasma cholesterol levels. This finding indicates that liver-derived plasma apoE can influence early atherogenesis through mechanisms other than modulation of lipoprotein metabolism and that liver-directed gene transfer and overexpression of apoE may be a therapeutic approach to atherosclerosis.