Purpose: To evaluate serum 25-hydroxyvitamin D levels in patients with keratoconus (KC) and its association with disease severity and to compare vitamin D levels with those of age- and sex-matched healthy individuals.Methods: This study included 100 patients with KC and 100 non-atopic healthy controls. Serum vitamin D levels were measured using liquid chromatography-tandem mass spectrometry and compared between the study groups. KC severity was graded as mild (K = 43.1-46.9 D), moderate (K = 47-50 D), and severe (K > 50 D). Vitamin D levels were classified as deficient (≤10 ng/mL), insufficient (11-20 ng/mL), and optimal (>20 ng/mL).Results: The mean serum vitamin D level was significantly lower in the KC group than in the control group (12.18 ± 5.05 ng/mL vs 15.18 ± 5.81 ng/mL; p < .001). Vitamin D deficiency (≤10 ng/mL) was observed in 54.0% of the KC patients and 34.0% of the controls (p = .011). Multivariable logistic regression analysis revealed that having deficient vitamin D level increased the presence of KC 2.9 fold (p = .021, 95% confidence interval, 1.17-6.95). Decreasing serum vitamin D levels were not significantly associated with increasing severity of KC (p = .311). Although the rate of patients with deficient vitamin D was the highest in the severe KC group, distribution of vitamin D levels were not significantly different among KC groups (p = .326).Conclusion: The patients with KC had lower serum vitamin D levels than those of age- and sex-matched healthy controls. The present study could be a valuable entry point for further researches investigating the potential link between vitamin D and KC.