ObjectiveTo investigate the causal relationship between gut microbiota and cholelithiasis using a two-sample Mendelian randomization method. MethodsThe genome-wide association studies (GWAS) data of gut microbiota from the MiBioGen study and the GWAS data of cholelithiasis from the FinnGen Biobank were utilized. Genetic variants significantly associated with the relative abundance of gut microbiota were identified as instrumental variables (IVs) based on a specified threshold. The inverse variance weighted (IVW) method was employed as the primary analytical approach, with results assessed based on the odds ratio (OR) and 95% confidence interval (CI). The robustness and reliability of the findings were ensured through quality control measures, including sensitivity analysis, assessment of heterogeneity, and evaluation for horizontal gene pleiotropy. ResultsClostridiumsensustricto1 [OR=1.160, 95%CI (1.023, 1.314), P=0.020], Coprococcus3 [OR=1.136, 95%CI (1.014, 1.272), P=0.028] and Peptococcus [OR=1.074, 95%CI (1.023, 1.128) , P=0.004] increased the risk of cholelithiasis. Bacilli [OR=0.897, 95%CI (0.818, 0.984), P=0.022], Family Ⅹ ⅢAD3011group [OR=0.908, 95%CI (0.830, 0.992), P=0.033] and Lactobacillales [OR=0.884, 95%CI (0.802, 0.974), P=0.013] were protective factors for cholelithiasis. ConclusionThe study has identified 6 kinds of specific gut microbiota that are causally linked to the development of cholelithiasis, providing new ideas for the diagnosis and treatment of cholelithiasis.