BACKGROUND: There was some evidence that gut microbiota was closely related to cholelithiasis, but the causal relationship between them remained unclear. In this study, we try to use Two-sample Mendelian randomization (MR) to clarify the potential causal relationship between gut microbiota and cholelithiasis. METHODS: Summary Genome-Wide Association Studies (GWAS) statistical data for gut microbiota was obtained from MiBioGen, and the data of cholelithiasis was obtained from UK Biobank (UKB). Two-sample MR analyses were performed to assess causalities between gut microbiota and cholelithiasis mainly using the inverse-variance weighted (IVW) method. Sensitivity analyses were used to determine the robustness of the MR results. Reverse MR analyses were performed to examine the reverse causal association. RESULTS: Our research results, based primarily on the IVW method, support the existence of a causal relationship between nine gut microbial taxa and cholelithiasis. We observed a positive association between G (p=0.032), (p=0.015), (p=0.003), (p=0.010) and cholelithiasis, while (p=0.031), (p=0.010), (p=0.036), (p=0.023), (p=0.022) may be associated with a reduced risk of cholelithiasis. We did not find a reverse causal relationship between cholelithiasis and 9 specific gut microbial taxa. CONCLUSIONS: This is the first mendelian randomization study to explore the causalities between specific gut microbiota taxa and cholelithiasis, which may provide new ideas and a theoretical basis for the prevention and treatment of cholelithiasis in the future.