Background: The association between vitamin D levels and non-alcoholic fatty liver disease (NAFLD) has been increasingly determined in recent researches. The aim of this study was to determine the association between vitamin D levels, measured as serum 25-hydroxy vitamin D (25(OH)D) and NAFLD. Serum 25(OH)D was prospectively determined in 80 patients. Of them, 40 subjects had NAFLD, whereas 40 subjects served as healthy control. Vitamin D deficiency was defined as serum 25(OH)D level < 20 ng/ml. Receiver operating characteristic (ROC) and regression analysis methods were used in our analysis. Results: About 70% of patients with NAFLD had vitamin D deficiency, but only 35% in the control group had. The mean serum 25 (OH)D was significantly lower in patients with NAFLD than the healthy control group (16.13 ± 10.23 versus 27.35 ± 10.58 ng/mL; P < 0.001). ROC curve analysis revealed that serum 25(OH)D level of less than 11.2 ng/ml increases the risk of NAFLD with 45% sensitivity and 97.5% specificity (Serum 25(OH)D level ≤ 18.1 ng/ml in males and ≤ 9.4 ng/ml in females increase the risk of NAFLD). Multivariate regression analysis showed that vitamin D deficiency, high age, and high BMI were associated with a significant high risk of NAFLD. Conclusion: NAFLD patients have low serum vitamin D concentrations, suggesting that vitamin D may have a role in the development of NAFLD. Future studies are recommended to determine the important therapeutic implications of vitamin D for the prophylaxis or the treatment of NAFLD.