The prevalence of hypovitaminosis D is high among obese subjects. Further, low 25-hydroxyvitamin D (25(OH)D) concentration has been postulated to be a risk factor for type 2 diabetes, although its relation with insulin-sensitivity is not well investigated. Thus, we aimed to investigate the relationship between 25(OH)D concentration and insulin-sensitivity, using the glucose clamp technique. In total, 39 subjects with no known history of diabetes mellitus were recruited. The association of 25(OH)D concentration with insulin-sensitivity was evaluated by hyperinsulinemic euglycemic clamp. Subjects with low 25(OH)D (<50 nmol/l) had higher BMI (P = 0.048), parathyroid hormone (PTH) (P = 0.040), total cholesterol (P = 0.012), low-density lipoprotein (LDL) cholesterol (P = 0.044), triglycerides (P = 0.048), and lower insulin-sensitivity as evaluated by clamp study (P = 0.047). There was significant correlation between 25(OH)D and BMI (r = -0.58; P = 0.01), PTH (r = -0.44; P < 0.01), insulin-sensitivity (r = 0.43; P < 0.01), total (r = -0.34; P = 0.030) and LDL (r = -0.40; P = 0.023) (but not high-density lipoprotein (HDL)) cholesterol, and triglycerides (r = 0.45; P = 0.01). Multivariate analysis using 25(OH)D concentration, BMI, insulin-sensitivity, HDL cholesterol, LDL cholesterol, total cholesterol, and triglycerides, as the cofactors was performed. BMI was found to be the most powerful predictor of 25(OH)D concentration (r = -0.52; P < 0.01), whereas insulin-sensitivity was not significant. Our study suggested that there is no cause-effect relationship between vitamin D and insulin-sensitivity. In obesity, both low 25(OH)D concentration and insulin-resistance appear to be dependent on the increased body size.