OBJECTIVE Microsomal triglyceride transfer protein (MTTP) is critical for the production of very-low-density lipoproteins (VLDL). The current studies were undertaken to examine the in vivo role of MTTP in hepatic cholesterol and fatty acid metabolism, as well as in biliary lipid secretion. We also tested whether MTTP plays a role in diet-induced cholelithiasis in mice. METHODS We used mice in which Mttp had been inactivated in the liver (Mttp(Delta/Delta) mice). We measured several parameters of cholesterol metabolism, fatty acid synthesis, and biliary lipid levels in mice fed a normal or a lithogenic diet. We also assessed the incidence of diet-associated gallstones. RESULTS Hepatic Mttp inactivation markedly decreased plasma triglyceride and cholesterol levels and increased biliary cholesterol and bile acid output. Hepatic cholesterogenesis and fatty acid synthesis were significantly decreased in Mttp(Delta/Delta) mice compared with control mice. The incidence of gallstones decreased from 90% in control mice to 33% in Mttp(Delta/Delta) mice after 8 weeks of a lithogenic diet (P < .0001). The mechanism of the protective effect appears to be increased biliary phospholipid output in Mttp(Delta/Delta) mice, leading to significant unsaturation of gallbladder bile. CONCLUSIONS These results indicate that modulation of Mttp expression in the liver affects hepatic lipid synthesis and storage as well as biliary lipid secretion. Our findings further indicate that inhibition of hepatic MTTP activity decreases the risk of experimental cholelithiasis by favoring phospholipid output into the bile.