Recent studies suggest an association of vitamin D with obesity, diabetes and cardiovascular diseases. We analyzed the association of serum vitamin D level assessed by 25-hydroxyvitamin D3 {25(OH)D3 } with nonalcoholic fatty liver disease (NAFLD) in apparently healthy men. We performed a cross-sectional study of 6,567 Korean men who participated in a health screening program, evaluating the association of serum 25(OH)D3 levels with the risk of NAFLD assessed by abdominal ultrasonogram. Of the participants, 43.6% had NAFLD and 21.1% had metabolic syndrome. Age, serum calcium, and aspartate aminotransferase levels showed weak but significant positive correlations with 25(OH)D3 level; total cholesterol, triglycerides, low-density lipoprotein cholesterol and fasting insulin level showed weak but significant negative correlations with 25(OH)D3 level. The mean 25(OH)D3 level was significantly lower in participants with NAFLD than in those without (38.7 ± 9.0 vs. 39.7 ± 9.7 nmol/L, p < 0.001). When participants were divided into tertiles based on mean 25(OH)D3 level, the proportion with NAFLD significantly increased as mean 25(OH)D3 level decreased (40.0, 45.0 and 45.9%, p for linear trend < 0.001). Multiple logistic regression analyses with NAFLD as the dependent variable showed that the tertiles with lower 25(OH)D3 levels had a significantly increased risk for NAFLD compared with the highest tertile, even after adjusting for body mass index and metabolic syndrome (OR 1.247 and 1.408 vs. the highest tertile, p < 0.001). Thus, participants with higher serum 25(OH)D3 showed a significantly reduced risk for NAFLD compared with the low 25(OH)D3 groups, independent of obesity and metabolic syndrome.