BACKGROUND: Vitamin D deficiency has reached epidemic proportions worldwide and has recently been linked to cardiometabolic risk factors including obesity, insulin resistance, hypertension, dyslipidemia as well as type 2 diabetes and cardiovascular disease. OBJECTIVE: The objective of this study was to examine the associations between circulating vitamin D levels and cardiometabolic risk factors using direct measures of adiposity, glucose intolerance, and insulin resistance, as well as lipids, blood pressure, and plasma markers of inflammation. METHODS: We measured circulating 25-hydroxyvitamin D3; physical activity (International Physical Activity Questionnaire); anthropometry (BMI, WHR, %body fat (dual energy X-ray absorptiometry)); metabolic parameters (fasting and 2-hour glucose during OGTT; insulin sensitivity (M, hyperinsulinaemic-euglycaemic clamp), and cardiovascular and inflammatory profiles (blood pressure (BP), lipids, white blood cell count (WBC), and plasma high-sensitivity C-reactive protein levels (hsCRP)) in 111 healthy, non-diabetic adults (66M/45F; age=31.1±9.2 years; %body fat=36.0±10.2%). RESULTS: Mean 25OHD was 39.8±19.8 nmol/L with no gender differences (p=0.4). On univariate analysis, vitamin D was associated with %body fat (r=-0.27;p=0.005), 2-hour glucose (r=-0.21;p=0.03), and insulin sensitivity (r=0.20;p=0.04), but not with age, BMI, WHR, fasting glucose, BP, lipids, or inflammatory markers (all p>0.05). After adjusting for age and sex, vitamin D remained associated with %body fat (β=-0.24;R2=0.38;p=0.003), 2-hour glucose (β=-0.22;R2=0.06;p=0.02), and insulin sensitivity (β=0.21;R2=0.05;p=0.03), and became associated with fasting glucose (β=-0.19;R2=0.10;p=0.04) and hsCRP (β=-0.21;R2= 0.12;p=0.04). After adjusting for age, sex, and %body fat, vitamin D was only associated with fasting glucose (β=-0.21;R2= 0.11;p=0.03), which persisted after hsCRP and physical activity were added to the model (β=-0.24;R2=0.15;p=0.02). CONCLUSION: These cross-sectional data suggest that associations between vitamin D and glucose metabolism among healthy non-diabetic adults are largely mediated by adiposity with no association with cardiovascular risk factors. Large-scale intervention and mechanistic studies are needed to further investigate whether vitamin D has an independent role in the prevention and/or management of cardiometabolic risk and disease.