Vegetarian diets may lower symptomatic gallstone disease via cholesterol lowering. This study aimed to examine the risk of symptomatic gallstone disease (GSD) in Taiwanese vegetarians vs. nonvegetarians in a prospective cohort and to explore if this association is related to cholesterol concentration. We prospectively followed 4839 participants, and in the 29,295 person-years of follow-up, 104 new incident GSD cases were confirmed. Diet was assessed through a validated food frequency questionnaire. Symptomatic GSD was ascertained through linkage to the Taiwan National Health Insurance Research Database. Blood cholesterol profiles were measured at recruitment. Cox regression was applied to assess the effect of diet on symptomatic GSD, adjusting for age, education, smoking, alcohol, physical activities, diabetes, kidney diseases, body mass index, lipid-lowering medication, and hypercholesterolemia. Vegetarian diet was associated with a decreased risk of symptomatic GSD compared with nonvegetarian diet in women (hazard ratio [HR], 0.52; 95% confidence interval [CI], 0.28⁻0.96) but not in men. In women, nonvegetarians with hypercholesterolemia had 3.8 times the risk of GSD compared with vegetarians with normal cholesterol (HR, 3.81, 95% CI, 1.61⁻9.01). A vegetarian diet may therefore protect against GSD independent of baseline hypercholesterolemia. A nonvegetarian diet and hypercholesterolemia may have an additive effect in increasing GSD risk in women.