In a long-term follow-up study of middle-aged men, the relation between the intake of energy, nutrients, and foods and the 25-year incidence of clinically diagnosed gallstones was studied. Information on the presence of gallstones was obtained by self-report and verified through medical records after death. Of 860 men, 54 developed symptomatic gallstones, yielding an incidence rate of 3.1/1000 person-years. The present study provides a comprehensive picture of dietary risk factors for clinically diagnosed gallstones based on a long-term follow-up. Calcium intake was inversely associated with gallstone incidence in the univariate and multivariate Cox proportional hazards analyses (hazard ratio (HR) upper tertile: 0.3; 95% CI: 0.1 to 0.7). A positive association with sugars (monosaccharides and disaccharides) appeared after the introduction of age, body mass index, calcium intake, and the intake of energy from nutrients other than sugars into the model (HR upper tertile: 2.3; 95% CI: 1.0 to 4.8). Calcium may alter the composition of bile by preventing the reabsorption of secondary bile acids in the colon, whereas sugars may influence bile composition through lipoprotein metabolism.