A strong association between the proportion of indigenous South American Mapuche ancestry and the risk of gallbladder cancer (GBC) has been reported in observational studies. Chileans show the highest incidence of GBC worldwide, and the Mapuche are the largest indigenous people in Chile. We set out to assess the confounding-free effect of the individual proportion of Mapuche ancestry on GBC risk and to investigate the mediating effects of gallstone disease and body mass index (BMI) on this association. Genetic markers of Mapuche ancestry were selected based on the informativeness for assignment measure, and then used as instrumental variables in two-sample Mendelian randomization analyses and complementary sensitivity analyses. Results suggested a putatively causal effect of Mapuche ancestry on GBC risk (inverse variance-weighted (IVW) risk increase of 0.8% per 1% increase in Mapuche ancestry proportion, 95% CI 0.4% to 1.2%, p = 6.7 × 10−5) and also on gallstone disease (3.6% IVW risk increase, 95% CI 3.1% to 4.0%), pointing to a mediating effect of gallstones on the association between Mapuche ancestry and GBC. In contrast, the proportion of Mapuche ancestry showed a negative effect on BMI (IVW estimate −0.006 kg/m2, 95% CI −0.009 to −0.003). The results presented here may have significant implications for GBC prevention and are important for future admixture mapping studies. Given that the association between the individual proportion of Mapuche ancestry and GBC risk previously noted in observational studies appears to be free of confounding, primary and secondary prevention strategies that consider genetic ancestry could be particularly efficient. © 2023 by the authors.Biobank of the University of Chile, (phs000810.v1.p1); National Heart, Lung, and Blood Institute, NHLBI; University of Miami, UM, (N01-HC65234); Northwestern University, NU, (N01-HC65236); San Diego State University, SDSU, (N01-HC65237); Albert Einstein College of Medicine, Yeshiva University, AECOM, (N01-HC65235); University of North Carolina Wilmington, UNCW, (N01-HC65233); Horizon 2020 Framework Programme, H2020, (825741); Deutsche Forschungsgemeinschaft, DFG, (INST 35/1314-1 FUGG, INST 35/1503-1 FUGG); Ministerium für Wissenschaft, Forschung und Kunst Baden-Württemberg, MWK