In addition to its pivotal role in psychosocial behavior, the hypothalamic neuropeptide oxytocin contributes to metabolic control by suppressing eating behavior. Its involvement in glucose homeostasis is less clear, although pilot experiments suggest that oxytocin improves glucose homeostasis. We assessed the effect of intranasal oxytocin (24 IU) administered to 29 healthy, fasted male subjects on glucose homeostasis measured by means of an oral glucose tolerance test. Parameters of glucose metabolism were analyzed according to the oral minimal model. Oxytocin attenuated the peak excursion of plasma glucose and augmented the early increases in insulin and C-peptide concentrations in response to the glucose challenge, while slightly blunting insulin and C-peptide peaks. Oral minimal model analyses revealed that oxytocin compared to placebo induced a pronounced increase in beta-cell responsivity (PHItotal) that was largely due to an enhanced dynamic response (PHId), and a more than two-fold improvement in glucose tolerance (disposition index). ACTH, cortisol, glucagon and non-esterified fatty acid concentrations were not or only marginally affected. These results indicate that oxytocin plays a significant role in the acute regulation of glucose metabolism in healthy humans and render the oxytocin system a potential target of antidiabetic treatment.