This study investigates the association between gut microbiota and glioblastoma (GBM) using Mendelian randomization (MR). The authors utilized summary statistics from genome-wide association studies (GWAS) of 211 gut microbiota and GBM to assess the causal relationship. They applied Inverse Variance Weighted (IVW), MR-Egger, and Weighted Median (WM) methods to evaluate the causal association, and Cochran’s Q statistic to test for heterogeneity. The MR-Pleiotropic Residuals and Outliers (MR-PRESSO) method was used to identify and eliminate SNPs with horizontal pleiotropic outliers. Reverse MR was also conducted to assess the causal relationship between GBM and specific gut microbiota. The results suggest that nine gut microbiota remain stable after considering heterogeneity and sensitivity methods. Family *Peptostreptococcaceae* and genus *Eubacterium brachy* group were associated with an increased risk of GBM, while family *Ruminococcaceae*, genus *Anaerostipes*, genus *Faecalibacterium*, genus *Lachnospiraceae UCG004*, genus *Phascolarctobacterium*, genus *Prevotella7*, and genus *Streptococcus* were associated with a reduced risk. After Benjamini and Hochberg (BH) correction, family *Ruminococcaceae* was identified as playing a protective role against GBM (OR = 0.04, 95% CI: 0.01–0.19, FDR = 0.003). The study concludes that family *Ruminococcaceae* is significantly associated with a reduced risk of GBM, highlighting its potential clinical significance in treating this aggressive brain tumor. The findings provide insights into the gut-brain axis and the role of gut microbiota in GBM development, suggesting that modulation of gut microbiota could be a promising therapeutic approach.This study investigates the association between gut microbiota and glioblastoma (GBM) using Mendelian randomization (MR). The authors utilized summary statistics from genome-wide association studies (GWAS) of 211 gut microbiota and GBM to assess the causal relationship. They applied Inverse Variance Weighted (IVW), MR-Egger, and Weighted Median (WM) methods to evaluate the causal association, and Cochran’s Q statistic to test for heterogeneity. The MR-Pleiotropic Residuals and Outliers (MR-PRESSO) method was used to identify and eliminate SNPs with horizontal pleiotropic outliers. Reverse MR was also conducted to assess the causal relationship between GBM and specific gut microbiota. The results suggest that nine gut microbiota remain stable after considering heterogeneity and sensitivity methods. Family *Peptostreptococcaceae* and genus *Eubacterium brachy* group were associated with an increased risk of GBM, while family *Ruminococcaceae*, genus *Anaerostipes*, genus *Faecalibacterium*, genus *Lachnospiraceae UCG004*, genus *Phascolarctobacterium*, genus *Prevotella7*, and genus *Streptococcus* were associated with a reduced risk. After Benjamini and Hochberg (BH) correction, family *Ruminococcaceae* was identified as playing a protective role against GBM (OR = 0.04, 95% CI: 0.01–0.19, FDR = 0.003). The study concludes that family *Ruminococcaceae* is significantly associated with a reduced risk of GBM, highlighting its potential clinical significance in treating this aggressive brain tumor. The findings provide insights into the gut-brain axis and the role of gut microbiota in GBM development, suggesting that modulation of gut microbiota could be a promising therapeutic approach.