Key points• Peristaltic function of the small intestine is compromised in cholelithogenic humans and in animal models of cholesterol gallstones.• In a mouse gallstone model fed a cholesterol-and cholic acid-enriched diet, we show that slowing of small intestinal transit is due to absorption of excess cholesterol molecules from the upper small intestine followed by their incorporation into sarcolemmal membranes of smooth muscle cells.• Blocking cholesterol absorption with ezetimibe (Zetia), an inhibitor of intestinal sterol transport, prevents cholesterol enrichment of sarcolemmal membranes and normalizes the motility disorder.• Although the primary source of intestinal cholesterol in mice is the lithogenic diet, in most cholesterol gallstone-prone humans, the small intestine is flooded continuously with an abundance of liver-secreted cholesterol molecules via bile.• Our findings imply that small intestinal hypomotility will amplify the cholelithogenic state because of hyperabsorption of cholesterol and 'secondary' bile salt synthesis by the gut's anaerobic microbiota.Abstract Cholesterol gallstones are associated with slow intestinal transit in humans as well as in animal models, but the molecular mechanism is unknown. We investigated in C57L/J mice whether the components of a lithogenic diet (LD; 1.0% cholesterol, 0.5% cholic acid and 17% triglycerides), as well as distal intestinal infection with Helicobacter hepaticus, influence small intestinal transit time. By quantifying the distribution of 3 H-sitostanol along the length of the small intestine following intraduodenal instillation, we observed that, in both sexes, the geometric centre (dimensionless) was retarded significantly (P < 0.05) by LD but not slowed further by helicobacter infection (males, 9.4 ± 0.5 (uninfected), 9.6 ± 0.5 (infected) on LD compared with 12.5 ± 0.4 and 11.4 ± 0.5 on chow). The effect of the LD was reproduced only by the binary combination of cholesterol and cholic acid. We inferred that the LD-induced cholesterol enrichment of the sarcolemmae of intestinal smooth muscle cells produced hypomotility from signal-transduction decoupling of cholecystokinin (CCK), a physiological agonist for small intestinal propulsion in mice. Treatment with ezetimibe in an amount sufficient to block intestinal cholesterol absorption caused small intestinal transit time to return to normal. In most cholesterol gallstone-prone humans, lithogenic bile carries large quantities of hepatic cholesterol into the upper small intestine continuously, thereby reproducing this dietary effect in mice.