Aim: Vitamin D deficiency has been proposed to be involved in obesity-induced metabolic disease. However, data on the relationship between 25-hydroxycholecalciferol (25(OH)D) and insulin resistance have been inconsistent, and few studies have investigated the active vitamin D metabolite, 1,25-dihydroxycholecalciferol (1,25(OH)2D). This study aimed to determine the relationship between circulating levels of both 25(OH)D and 1,25(OH)2D and direct measures of glucose metabolism and insulin action in obese women. Methods: Serum levels of 25(OH)D and 1,25(OH)2D, and glucose metabolism and tissue-specific insulin action, as assessed in the basal state and during a two-step euglycaemic-hyperinsulinaemic clamp study with [6,6-2H2]glucose infusion, were measured in 37 morbidly obese women (age: 43±10 years; body mass index: 44±6kg/m2). Results: Sixteen subjects had circulating 25(OH)D levels<50nmol/L, consistent with vitamin D deficiency, and 21 had normal 25(OH)D levels. There were no differences in either baseline characteristics or parameters of glucose metabolism and insulin action between the groups. Serum 25(OH)D, but not 1,25(OH)2D, was negatively correlated with both body mass index (r =-0.42, P =0.01) and total body fat (r =-0.46, P <0.01). Neither 25(OH)D nor 1,25(OH)2D levels were related to any measured metabolic parameters, including fasting glucose, fasting insulin, basal endogenous glucose production, and hepatic, adipose-tissue and skeletal muscle insulin sensitivity. Conclusion: Obesity was associated with lower levels of circulating 25(OH)D, but not with the hormonally active metabolite 1,25(OH)2D. Neither 25(OH)D nor 1,25(OH)2D were related to glucose metabolism and tissue-specific insulin sensitivity in obese women, suggesting that vitamin D does not play a major role in obesity-related insulin resistance.