Background <p>While observational studies have suggested associations between gut microbiota composition, metabolomic alterations, and endometrial cancer risk, the causal nature of these relationships remains unclear due to potential confounding factors and reverse causation.</p> Methods <p>We<?tk 2?> conducted a comprehensive two-sample Mendelian randomization analysis using genetic instrumental variables derived from the largest available genome-wide association studies. We investigated causal relationships between specific gut microbiota taxa (Alcaligenaceae family, Ruminococcaceae NK4A214 and UCG014 groups), over 40 circulating metabolites spanning diverse biochemical pathways (including amino acids, lipids, vitamins, and fatty acids), and endometrial cancer risk.<?tk 0?></p> Results <p>No significant causal associations were identified between any of the examined gut microbiota taxa and endometrial cancer risk. All 95% confidence intervals crossed the null effect line (OR = 1.0) across different analytical methods. Similarly, comprehensive metabolomic analysis revealed no statistically significant causal relationships between circulating metabolite levels and endometrial cancer risk, including specific investigation of dodecadienoate, a polyunsaturated fatty acid derivative. Multi-method validation consistently demonstrated effect estimates close to zero with overlapping confidence intervals, indicating robust null findings across all analytical approaches.</p> Conclusions <p> This large-scale Mendelian randomization study provides strong genetic evidence against direct causal relationships between gut microbiota composition, circulating metabolomic profiles, and endometrial cancer risk.</p>