Purpose: We performed this study to determine the association between serum 25-hydroxyvitamin D [25(OH) D] level and myopia in adults. Methods: A total of 25,199 subjects aged >= 20 years were included from the National Health and Nutrition Examination Survey 2008-2012. Blood 25(OH)D levels were evaluated from blood samples. Refractive error was measured without cycloplegia. Myopia and high myopia were defined as >=-0.50 diopters (D) and >=-6.0 D, respectively. Other covariates such as education, physical activity, and economic status were obtained from interviews. Results: Linear regression analysis showed that as 25(OH) D level increased by 1 ng/mL, myopic refractive error significantly decreased by 0.01 D (P < 0.001) after adjusting for potential confounders including sex, age, height, education level, economic status, physical activity, and sunlight exposure time. The odds ratios for myopia was 0.75 (95% Confidence interval [CI]; 0.67-0.84, P < 0.001) in the highest 25(OH) D quintile compared to the lowest quintile. The odds ratios for high myopia was 0.63 (95% CI; 0.47-0.85, P < 0.001) in the highest 25(OH)D quintile compared to the lowest quintile. Conclusion: Serum 25(OH)D level was inversely associated with myopia in Korean adults.