Transporters present in the epithelium of the small intestine determine the efficiency by which dietary and biliary cholesterol are taken up into the body and thus control whole-body cholesterol balance. Niemann-Pick C1 Like Protein 1 (Npc1l1) transports cholesterol into the enterocyte, whereas ATP-binding cassette transporters Abca1 and Abcg5/Abcg8 are presumed to be involved in cholesterol efflux from the enterocyte towards plasma HDL and back into the intestinal lumen, respectively. Abca1, Abcg5, and Abcg8 are well-established LXR target genes. We examined the effects of a high-fat diet on expression and function of cholesterol transporters in the small intestine in mice. Npc1l1, Abca1, Abcg5, and Abcg8 were all down-regulated after 2, 4, and 8 weeks on a cholesterol-free, high-fat diet. The high-fat diet did not affect biliary cholesterol secretion but diminished fractional cholesterol absorption from 61% to 42% (p<0.05). In an acute experiment, in which triacylglycerols of unsaturated fatty acids were given by gavage, we found that this down-regulation occurs within a 6 hours time frame. Studies in LXR-null mice, confirmed by in vitro data, showed that fatty acid-induced down-regulation of cholesterol transporters is LXR-independent and associated with a post-translational increase in Hmg-CoA reductase activity that reflects induction of cholesterol biosynthesis as well as with a doubling of neutral fecal sterol loss. This study highlights the induction of adaptive changes in small intestinal cholesterol metabolism during exposure to dietary fat.