BACKGROUND: The role of gut microbiota in tumorigenesis has gained significant attention in recent years. This study aimed to investigate the causal relationship between gut microbiota and three types of urothelial carcinoma (renal pelvic cancer [RPC], urothelial carcinoma [UC], and bladder cancer [BC]), and to explore potential underlying mechanisms. METHODS: Two-sample Mendelian randomization (MR) analysis was conducted using GWAS data to identify gut microbiota-associated SNPs and evaluate their causal effects on the three cancers. Sensitivity analysis, reverse MR analysis, and mediation MR analysis were further performed to examine the roles of gut microbiota, inflammatory factors, and blood cells in cancer development. RESULTS: Mediation MR analysis revealed that Bacillaceae A significantly increased RPC risk by elevating IL-6 levels; CAG-452 promoted RPC by reducing SCF levels; and Succiniclasticum increased RPC risk by lowering platelet reactivity (WNR_Isobutyric_A._PLT_SD_SFL) (mediation effect ratio: 34.01%). Conversely, UCG-010 sp003150215 reduced RPC risk by increasing platelet reactivity (mediation effect ratio: 30.51%). For UC, Syntrophorhabdaceae promoted UC development by lowering IL-4 levels (mediation effect ratio: 22.46%), while Prevotella sp002437285 increased UC risk through modulation of WNR_Captopril_PLT_SD_SSC (mediation effect ratio: 30.58%). CONCLUSION: This research identifies causal relationships between gut microbiota and RPC, UC, and BC, and elucidates their impact on cancer development through mediation by inflammatory factors and blood cells. These findings provide new perspectives and a foundation for the potential application of gut microbiota in cancer prevention and treatment.