Gallstones form when the ratio of bile cholesterol to bile acids and phospholipids is elevated, causing cholesterol to precipitate. Physical inactivity is hypothesized to increase gallstone development, but experimental evidence supporting this is lacking, and potential mechanisms for the anti-lithogenic effects of exercise have not been described. The purpose of this study was to examine the effect of endurance exercise training on gallstone formation and the expression of genes involved in bile cholesterol metabolism in gallstone-sensitive (C57L/J) mice. At 10 weeks, 50 male mice began a lithogenic diet and were randomly assigned to an exercise-training (EX) or sedentary (SED) group (n=25 per group). Mice in the EX group ran on a treadmill at ~15m/min for 45 minutes/day for 12 weeks. At sacrifice, gallstones were collected, pooled by group, and weighed. The weight of the gallstones was 2.5-fold greater in the SED mice compared to EX (143 mg vs. 57 mg, respectively). In the EX mice, hepatic expression of the LDLr, SRB1, and Cyp27 was increased by ~2-fold (p < 0.05 for each). The LDLr and SRB1 increase cholesterol clearance by LDL and HDL particles, respectively, while Cyp27 promotes the catabolism of cholesterol to bile acids. Taken together, these data indicate that exercise promotes changes in hepatic gene expression that increase cholesterol uptake by the liver, but simultaneously increase the catabolism of cholesterol to bile acids, effectively reducing cholesterol saturation in the bile. This suggests a mechanism by which exercise improves cholesterol clearance from the circulation while simultaneously inhibiting gallstone formation.