This study aimed to investigate the association between folate levels and the prevalence of glaucoma. This nationwide population-based cross-sectional study included 1790 participants aged ≥40 years. We analyzed data regarding the participants obtained in the 2016-2018 Korean National Health and Nutrition Examination Survey. The diagnosis of glaucoma was defined according to the International Society of Geographical and Epidemiological Ophthalmology criteria. Logistic regression analyses were used to investigate the relationship between blood folate levels and glaucoma. There was a significantly lower prevalence of glaucoma in the highest quartile of blood folate levels than in the lowest quartile, after adjusting for confounding factors such as age, sex, systemic hypertension, diabetes, hypercholesterolemia, and smoking (odds ratio [OR] = 0.470; 95% confidence interval [CI], 0.291-0.759; P for trend = 0.017). There was a significantly lower risk of glaucoma in the highest quartile of blood folate levels than in the lowest quartile among women (OR = 0.188; 95% CI, 0.099-0.357; P for trend <0.001) and younger participants (OR =0.443; 95% CI, 0.229-0.856; P for trend = 0.045). Our findings indicate a strong inverse correlation between blood folate levels and glaucoma.