PURPOSE: The purpose of this study is to explore the causal relationship between gut microbiota and gastrointestinal (GI) cancers and to investigate the potential mediating factors influencing the development of GI cancers. METHODS: Using data from genome-wide association studies (GWAS), we employed two-sample Mendelian randomization (TSMR) to explore the relationship among gut microbiota, inflammatory cytokines and GI cancers. Subsequently, a multivariable Mendelian randomization (MVMR) analysis was meticulously conducted to perform a mediation analysis, thereby estimating the proportion of mediation effects conferred by inflammatory cytokines. RESULTS: TSMR analysis established a causal relationship between 23 gut microbiota taxa and 11 inflammatory cytokines with GI cancers. Specifically, 7 gut microbiota taxa were associated with an increased risk of gastric cancer (GC), 6 with small intestine cancer, and 10 with colorectal cancer (CRC). Among the inflammatory cytokines, 4 were linked to GC risk, 3 to small intestine cancer, and to CRC. Mediation analysis further indicatedthat tumor necrosis factor ligand superfamily member 12 (TNFSF12) mediated 9.703% (95% CI 0.108%~15.891%) of the total effect of genus Ruminiclostridium9 on GC. CONCLUSION: Our findings support a causal relationship between gut microbiota, inflammatory cytokines, and GI cancers. These biomarkers provide new insights into the mechanisms underlying GI cancers and have the potential to improve strategies forprevention, diagnosis, and treatment.