Mechanisms to increase reverse cholesterol transport (RCT) and biliary sterol disposal are currently sought to prevent atherosclerosis. Previous work with HepG2 cells and primary hepatocytes showed that carboxyl ester lipase (CEL), a broad-spectrum lipase secreted by pancreas and liver, plays an important role in hydrolysis of high-density lipoprotein (HDL) cholesteryl esters (CEs) after selective uptake by hepatocytes. The effect of CEL on RCT of HDL cholesterol was assessed by measuring biliary and fecal disposal of radiolabeled HDL-CE in control and Cel(-/-) mice. Radiolabeled CE was increased 3-fold in hepatic bile of Cel(-/-) mice, and the mass of CE in gall bladder bile was elevated. Total radiolabeled transport from plasma to hepatic bile was more rapid in Cel(-/-) mice. Fecal disposal of radiolabel from HDL-CE, as well as total sterol mass, was markedly elevated for Cel(-/-) mice, primarily due to more CE. RCT of macrophage CE was also increased in Cel(-/-) mice, as measured by excretion of radiolabel from injected J774 cells. Increased sterol loss was compensated by increased cholesterol synthesis in Cel(-/-) mice. Together, the data demonstrate significantly increased RCT in the absence of CEL and suggest a novel mechanism by which to manipulate plasma cholesterol flux.