It has been found that polymorphisms in the apolipoprotein (APO)-B gene are associated with cholesterol gallstones in humans. We hypothesized that APO-B plays a major regulatory role in the response of biliary cholesterol secretion to high dietary cholesterol and contributes to cholesterol gallstone formation. In the present study, we investigated whether lack of expression of intestinal Apob48 or Apob100 reduces susceptibility to cholesterol gallstones by decreasing intestinal absorption and biliary secretion of cholesterol in male mice homozygous for an “APO-B48 only” allele (Apob48/48), an “APO-B100 only” allele (Apob100/100), or a wild-type APO-B allele (Apob+/+) before and during an 8-week lithogenic diet. We found that cholesterol absorption was significantly decreased as a result of the APO-B48 deficiency in Apob100/100 mice compared with wild-type and Apob48/48 mice, regardless of whether chow or the lithogenic diet was administered. Consequently, hepatic cholesterol synthesis was significantly increased in Apob100/100 mice compared with wild-type and Apob48/48 mice. On chow, the APO-B100 deficiency in Apob48/48 mice with reduced plasma levels of LDL/VLDL —but not HDL cholesterol—induced relative hyposecretion of biliary bile salts and phospholipids accompanying normal biliary cholesterol secretion. Compared with Apob48/48 and wild-type mice, lithogenic diet–fed Apob100/100 mice displayed significantly lower secretion rates of biliary cholesterol, but not phospholipid or bile salts, which results in significant decreases in prevalence rates, numbers, and sizes of gallstones. In conclusion, absence of expression of intestinal Apob48, but not Apob100, reduces biliary cholesterol secretion and cholelithogenesis, possibly by decreasing intestinal absorption and hepatic bioavailability. (HEPATOLOGY 2005;42:894–904.)