BACKGROUND & AIMS: Obesity, type 2 diabetes, and lifestyle factors (cigarette smoking, alcohol drinking, and coffee consumption) have been associated with the risk of developing gallstone disease in observational studies, but whether these associations are causal is undetermined. We conducted a Mendelian randomization study to assess these associations. METHODS: Genetic instruments associated with the exposures at the genome-wide significance (p < 5×10) level were selected from corresponding genome-wide association studies (n=224 459 to 1 232 091 individuals). Summary-level data for gallstone disease were obtained from the UK Biobank (10 520 cases and 350 674 non-cases) and FinnGen consortium (11 675 cases and 121 348 non-cases). Univariable and multivariable Mendelian randomization analyses were conducted. Results from UK Biobank and FinnGen were combined using fixed-effects meta-analysis. RESULTS: The odds ratios were 1.63 (95% confidence interval (CI), 1.49, 1.79) for one standard deviation (SD) increase in body mass index, 1.81 (95% CI, 1.60, 2.05) for one SD increase in waist circumference, 1.13 (95% CI, 1.09, 1.17) for one unit increase in the log-odds ratio of type 2 diabetes and 1.25 (95% CI, 1.16, 1.34) for one SD increase in prevalence of smoking initiation. The associations for body mass index and type 2 diabetes persisted after mutual adjustment. Genetically predicted coffee consumption was inversely associated with gallstone disease after adjustment for body mass index and smoking (odds ratio per 50% increase 0.44, 95% CI, 0.21, 0.91). There was no association with alcohol consumption. CONCLUSIONS: This study supports independent causal roles of obesity, type 2 diabetes, and smoking in gallstone disease.