BACKGROUND: Given the increasing interest in the role of gut microbiota in glioblastoma multiforme (GBM), our objective was to examine the potential causal relationship between gut microbiota and GBM, as well as the mediating effects of specific metabolites. METHODS: A bidirectional two-sample Mendelian randomization (MR) analysis was conducted to investigate the associations between 196 microbial taxa and GBM. A two-step MR technique was used to identify significant mediators in this relationship. Subsequently, a mediation analysis was performed to explore and quantify the mediating effects of specific metabolites on the causal relationship between gut microbiota and GBM. RESULTS: Five taxa showed significant associations with GBM. Among them, [odds ratio (OR): 1.95; 95% confidence interval (CI): 1.21, 3.13;  = 0.005] and (OR: 1.81; 95% CI: 1.04, 3.15;  = 0.036) were positively correlated with the risk of GBM, while had a protective effect against GBM (OR: 0.45; 95% CI: 0.22, 0.89;  = 0.021). The mediation analysis revealed that the connections among and GBM were mediated by Methyl-4-hydroxybenzoate sulfate, phosphoethanolamine and dehydroepiandrosterone sulfate. Each of these accounted for 7.27, 7.98, and 8.65%, respectively. CONCLUSION: Our study provides evidence supporting a potential causal association between certain gut microbiota taxa and GBM. The study highlights the central role of gut microbiota in GBM pathogenesis and their interactions with vital serum metabolites. This paves the way for potential novel therapeutic interventions in GBM management.