The identification of ABCA1 as a key transporter responsible for cellular lipid efflux has led to considerable interest in defining its role in cholesterol metabolism and atherosclerosis. In this study, the effect of overexpressing ABCA1 in the liver of LDLr-KO mice was investigated. Compared to LDLr-KO mice, ABCA1-Tg x LDLr-KO (ABCA1-Tg) mice had significantly increased plasma cholesterol levels, mostly due to a 2.8-fold increase in cholesterol associated with a large pool of apoB-lipoproteins. ApoB synthesis was unchanged but the catabolism of ¹²⁵I-apoB-VLDL and -LDL were significantly delayed, accounting for the 1.35-fold increase in plasma apoB levels in ABCA1-Tg mice. We also found rapid in vivo transfer of free cholesterol from HDL to apoB-lipoproteins in ABCA1-Tg mice, associated with a significant 2.7-fold increase in the LCAT-derived cholesteryl linoleate content found primarily in apoB-lipoproteins. ABCA1-Tg mice had 1.4-fold increased hepatic cholesterol concentrations, leading to a compensatory 71% decrease in de novo hepatic cholesterol synthesis, as well as enhanced biliary cholesterol and bile acid secretion. CAV-1, CYP2b10 and ABCG1 were significantly induced in ABCA1-overexpressing livers; however, no differences were observed in the hepatic expression of CYP7a1, CYP27A1 or ABCG5/G8 between ABCA1-Tg and control mice. As expected from the pro-atherogenic plasma lipid profile, aortic atherosclerosis was increased 10-fold in ABCA1-Tg mice. In summary, hepatic overexpression of ABCA1 in LDLr-KO mice leads to (1) expansion of the pro-atherogenic apoB-lipoprotein cholesterol pool size via enhanced transfer of HDL-cholesterol to apoB-lipoproteins and delayed catabolism of cholesterol-enriched apoB-lipoproteins, (2) increased cholesterol concentration in the liver, resulting in upregulated hepatobiliary sterol secretion and (3) significantly enhanced aortic atherosclerotic lesions.