OBJECTIVE To explore a hypothesized association between vitamin D inadequacy and diabetic retinopathy. METHODS This cross-sectional study analyzed data from individuals aged 40 years and older with diabetes mellitus who participated in the interview and medical examination components of the Third National Health and Nutrition Examination Survey conducted from October 1, 1988, through September 30, 1994. The relationship between diabetic retinopathy and serum 25-hydroxyvitamin D concentration was evaluated using regression analysis in the presence of demographic and clinical covariates, such as age, race, obesity, and persistent hyperglycemia. RESULTS On the basis of the 1790 adults with diabetes who met the study's inclusion criteria, the percentage of individuals with vitamin D deficiency increased with severity of retinopathy: no retinopathy, 27.9%; mild, 28.2%; moderate to severe, 43.2%; and proliferative, 64.6% (P = .01). Regression analysis of retinopathy severity vs serum 25-hydroxyvitamin D concentration did not demonstrate a statistically significant relationship between the two variables (P = .07). CONCLUSIONS This study found an association between severity of diabetic retinopathy and prevalence of vitamin D deficiency, but the findings were inconclusive about the existence of a relationship between retinopathy severity and serum 25-hydroxyvitamin D concentration. Given previous research indicating possible anti-inflammatory and antiangiogenic properties of vitamin D, the connection between vitamin D and diabetic retinopathy warrants further study.