Objective: We carried out a two-sample Mendelian Randomization (MR) analysis to investigate the potential causal relationship between gut microbiota (GM) and primary open-angle glaucoma (POAG). Methods: The exposure data was derived from the Genome-Wide Association Studies (GWAS) of GM at the University of Bristol, while the outcome data for POAG was sourced from the MRC Integrative Epidemiology Unit (IEU) Open GWAS database. In this study, we employed MR methods including Inverse Variance Weighted (IVW), MR Egger, Weighted Median (WM), Simple Mode, and Weighted Mode to investigate the potential causal relationships between GM and POAG. IVW was used as the primary method for this study. Sensitivity analysis was conducted to assess the reliability of the MR analysis. Results: The IVW analysis confirmed that Butyrivibrio (OR=1.170, 95% CI: 1.057~1.295, P=0.002), Howardella (OR=1.188, 95% CI: 1.043~1.355, P=0.010), and LachnospiraceaeUCG001 (OR=1.229, 95% CI: 1.016~1.485, P=0.033) were correlated with the risk of POAG. Conversely, Candidatus Soleaferrea (OR=0.810, 95% CI: 0.670~0.981, P=0.031), Ruminococcus torques (OR=0.656, 95% CI: 0.453~0.950, P=0.026), and Ruminococcaceae UCG013 (OR=0.770, 95% CI: 0.598~0.990, P=0.041) were protective factors for POAG. The heterogeneity and pleiotropy among the instrumental variables were assessed through sensitivity analysis. Conclusion: The MR study indicated a causal relationship between GM and POAG. Given the sight-threatening characteristic of POAG, early identification and intervention in the relative factors was significant for the prognosis of POAG. Keywords: Gut microbiota; Primary open-angle glaucoma; Mendelian Randomization