Several lines of evidence suggest that ovarian hormones influence glucose homeostasis, although their exact role in humans has not been clearly defined. We sought to test the hypothesis that ovarian hormones regulate glucose homeostasis by examining the effect of pharmacologically-induced ovarian hormone deficiency on glucose disposal and insulin secretion. Young, healthy women with regular menstrual patterns were studied during the follicular and luteal phases of their cycle at baseline and after two months of treatment with gonadotropin-releasing hormone agonist (GnRHa; n=7) or placebo (n=6). Using hyperglycemic clamps, in combination with stable isotope-labeled (i.e., ¹³C and ²H) glucose tracers, we measured glucose disposal and insulin secretion. Additionally, we assessed body composition and regional fat distribution using imaging techniques, as well as glucoregulatory hormones. Ovarian hormone suppression did not alter body composition, abdominal fat distribution or thigh tissue composition. There was no effect of ovarian suppression on total, oxidative or non-oxidative glucose disposal expressed relative to plasma insulin level. Similarly, no effect of ovarian hormone deficiency was observed on first- or second phase insulin secretion or insulin clearance. Finally, ovarian hormone deficiency was associated with an increase in circulating adiponectin levels, but no change in leptin concentration. Our findings suggest that a brief period of ovarian hormone deficiency in young, healthy eugonadal women does not alter glucose disposal index or insulin secretion, suggesting that ovarian hormones play a minimal role in regulating glucose homeostasis. Our data do, however, support a role for ovarian hormones in the regulation of plasma adiponectin levels.