Low levels of serum 25-hydroxy vitamin D (25(OH)D), have been associated with development of type 2 diabetes and cardiovascular disease (CVD); however there are limited data on serum 25(OH)D in Indigenous Australians, a population at high risk for both diabetes and CVD. We aimed to assess levels of serum 25(OH)D in Aboriginal and Torres Strait Islander Australians and to explore relationships between 25(OH)D and cardio-metabolic risk factors and diabetes. 592 Aboriginal and/or Torres Strait Islander Australian participants of The eGFR (estimated glomerular filtration rate) Study, a cross-sectional analysis of a cohort study performed in 2007-2011, from urban and remote centres within communities, primary care and tertiary hospitals across Northern Territory, Far North Queensland and Western Australia. Assessment of serum 25(OH)D, cardio-metabolic risk factors (central obesity, diabetes, hypertension, history of cardiovascular disease, current smoker, low HDL-cholesterol), and diabetes (by history or HbA1c ≥6.5%) was performed. Associations were explored between 25(OH)D and outcome measures of diabetes and number of cardio-metabolic risk factors. The median (IQR) serum 25(OH)D was 60 (45-77) nmol/L, 31% had 25(OH)D <50 nmol/L. For participants with 25(OH)D < 50 vs ≥50 nmol/L, cardio-metabolic risk profile differed for: diabetes (54%, 36% p < 0.001), past history of cardiovascular disease (16%, 9%, p = 0.014), waist-hip ratio (0.98, 0.92, p < 0.001), urine albumin-creatinine ratio (2.7, 1.5 mg/mmol, p < 0.001). The OR (95% CI) for diabetes was 2.02 (1.03 - 3.95) for people in the lowest vs highest tertiles of 25(OH)D (<53 vs >72 nmol/L, respectively) after adjusting for known cardio-metabolic risk factors. The percentage of 25(OH)D levels <50 nmol/L was high among Aboriginal and Torres Strait Islander Australians from Northern and Central Australia. Low 25(OH)D level was associated with adverse cardio-metabolic risk profile and was independently associated with diabetes. These findings require exploration in longitudinal studies.