Elevated intraocular pressure (EIOP) is the only major modifiable risk factor of glaucoma. While low serum vitamin D levels are considered a potential risk factor for glaucoma, there is conflicting evidence on the relationship between vitamin D and EIOP despite the possible linkage between vitamin D and intraocular pressure through oxidative stress and systemic inflammation. Therefore, the aim of this study is to verify the relationship between 25-hydroxyvitamin D [25(OH)D] and EIOP using data from 15,338 individuals who visited the health promotion center of an education hospital. The cubic spline curve revealed an inverse dose-dependent association between serum 25(OH)D level and EIOP. Using multiple logistic regression analysis, the fully adjusted odds ratio (OR) with 95% confidence interval (CI) for the EIOP of the serum 25(OH)D per increment was 0.97 (0.96–0.990). The fully adjusted ORs (95% CIs) for the EIOP of the 25(OH)D insufficiency and 25(OH)D sufficiency groups, compared to 25(OH)D deficiency group, were 0.72 (0.56–0.92) and 0.51 (0.34–0.78), respectively. The relationship remained significant in male and young age subgroups. In conclusion, the clinical assessment of intraocular pressure may prove helpful when treating patients with 25(OH)D deficiency, which may be a preventive strategy against the development of glaucoma.