Purpose Cheese is a dairy product with high calcium content. It has been suggested that calcium intake may increase fecal excretion of bile acids that would cause a regeneration of bile acids from hepatic cholesterol and thereby result in a lowering of plasma cholesterol concentrations. We aimed to test this hypothesis by assessing bile acid and calcium concentrations in fecal samples from humans after intake of cheese and butter. Methods The study was a randomized, 2 × 6 weeks crossover, dietary intervention study including 23 men and women who replaced part of their habitual dietary fat intake with 13% energy from cheese or butter. Results After 6 weeks of intervention cheese resulted in higher amounts of calcium excreted in feces compared to butter. However, no difference was observed in fecal bile acid output despite lower serum total, LDL and HDL cholesterol concentrations observed with cheese intake. Conclusion We were not able to confirm the hypothesis that calcium from cheese increases the excretion of fecal bile acids. Therefore, the mechanisms responsible for the lowering of cholesterol concentrations with cheese compared to butter intake remains unresolved.