ABSTRACT Background We investigated the effects of vitamin D on the ocular surface, tear functions, corneal imaging, and tear film cytokine levels. Methods Fifty-two patients with vitamin D levels were examined in 3 groups according to serum vitamin D levels; 28 in group 1 (<12 ng/ml), 10 in group 2 (12–20 ng/ml), and 14 in group 3 (>20 ng/ml). Ocular surface disease index (OSDI), tear break up time (BUT), lissamine green (LG) staining, Schirmer test, in vivo confocal microscopy (IVCM), and tear collection for cytokine analysis were performed. Results The mean OSDI score was 35.2 ± 23.3, 36.2 ± 17.7, 24.4 ± 18.2 (p = .253), TBUT was 6.7 ± 2.5 sec, 9.3 ± 1.8 sec, 11.1 ± 2.8 sec (p < .001), Schirmer test was 16.7 ± 8.5 mm, 18.7 ± 7.6 mm, and 20.2 ± 7 mm (p = .254), median LG staining grade was 1 (0–3), 1 (0–2), 0 (0–1) (p = .008) in group 1, group 2, and group 3, respectively. Basal epithelial cell density was 4 027 ± 512 cells/mm2, 4 673 ± 451 cells/mm2, 5 067 ± 817 cells/mm2 (p = < 0.001), sub-basal nerve density was 978 ± 204 μm/frame, 1 236 ± 172 μm/frame, 1 425 ± 290 μm/frame (p = <0.001), median number of long nerve fibers was 3 (2–4) nerve/frame, 4 (3–4) nerve/frame, 4 (3–6) nerve/frame (p = .001), and median grade of nerve fiber tortuosity was 2 (0–3), 2.5 (2–3), 3 (2–4) (p < .001) in group 1, group 2, and group 3, respectively. Mean IL-1 β (82.62 ± 15.26, 85.57 ± 17.41, and 66.44 ± 11 ng/ml in group 1, 2 and 3, respectively, p = .002), IL-17 (77.80 ± 24.91, 64.46 ± 25.47, 55.42 ± 12.05 ng/ml in group 1, 2 and 3, respectively, p = .012), and IL-2 (75.7 ± 18.4, 66.13 ± 26.78, and 59.65 ± 16.04 ng/ml in group 1, 2 and 3, respectively, p = .048) levels were significantly lower in group 3, whereas, IL-13 levels were significantly higher in group 3 (16.12 ± 5.24, 19.20 ± 4.90, and 21.6 ± 5.55 ng/ml in groups 1, 2, and 3, respectively, p = .010). Conclusions Vitamin D deficiency/insufficiency is associated with ocular surface changes shown with significant TBUT, LG staining, and tear film cytokine contents. Besides, significant corneal basal epithelial, sub-basal nerve density, and structural sub-basal nerve changes were associated with lower Vitamin D levels.