To study the correlation of serum vitamin D levels with quantitative (central subfield thickness [CST], cube average thickness [CAT]), cross-sectional (disorganisation of retinal inner layer [DRIL] and ellipsoid zone [EZ]) and topographic parameters (retinal pigment epithelium [RPE]) on spectral domain optical coherence tomography (SD-OCT) in diabetic retinopathy (DR), for the first time. Methods: Eighty-eight consecutive cases of type 2 diabetes mellitus with no retinopathy (No DR; n = 22); non-proliferative DR (NPDR; n = 22); proliferative DR (PDR; n = 22) and healthy controls (n = 22) were included, after sample size calculation. On SD-OCT, physician-friendly grading systems were created for DRIL, EZ disruption and RPE alterations. Serum vitamin D was analysed using a standard protocol. Statistical analysis was done using Pearson correlation, Student's t-test, ANOVA, Newman-Keuls test, chi-square test and univariate ordinal logistic regression analysis. Results: Mean serum vitamin D levels (ng/ml) were: No DR = 23.36 ± 2.00, NPDR = 17.88 ± 1.86, PDR = 14.07 ± 1.21, and controls = 25.11 ± 1.59. Low vitamin D levels correlated significantly with severity of retinopathy, VA (r = 0.50), CST (r = 0.36), CAT (r = 0.41), DRIL (r = 0.35), EZ disruption (r = 0.40) and RPE alterations (r = 0.37), respectively (p < 0.01). Significantly low vitamin D levels were observed in subjects with DRIL present versus DRIL absent; EZ disruption, focal versus global versus intact; RPE alterations, focal versus global versus none, respectively (p < 0.05). Conclusions: Low serum vitamin D levels correlate with the presence of DRIL, EZ disruption and RPE alterations and increased severity of DR.