Purpose: The purpose of this study is to evaluate the effect of vitamin D deficiency on corneal endothelial layer using specular microscopy. Methods: Fifty-eight eyes of 58 patients whose vitamin 13 level was below 15 ng/ml and who had no ocular pathology were included in the study (Group 1). Forty eyes of 40 age-and sex-matched subjects were enrolled as control group (Group 2). Corneal endothelial cell density (CD), coefficient of variation (CV), hexagonal cell ratio (HEX), and central corneal thickness (CCT) were measured using specular microscopy (Konan Medical Inc., Nishinomiya, Japan). The obtained data were compared between the groups. Results: There was no significant difference between the groups in terms of age and gender (P = 0.344, P = 0.399, respectively). The mean CD value was 2772.79 +/- 202.21 cells/mm(2) in Group 1 and 2954.97 +/- 116.89 cells/mm(2) in Group 2 (P = 0.001). The mean CV value was 30.31 +/- 3.65 in Group 1 and 28.20 +/- 2.71 in Group 2 (P = 0.003). The mean HEX value was 46.56 +/- 6.32 in Group 1 and 51.07 +/- 5.28 in Group 2 (P = 0.001). The mean CCT value was 555.87 +/- 36.90 mu in group 1 and 549.0 +/- 37.39 mu in Group 2 (P = 0.96). Conclusion: Vitamin D deficiency may affect the corneal endothelial layer. Patients with vitamin D deficiency should be evaluated for endothelial parameters in particular before an intraocular surgery. Further studies are needed to confirm our results.