Abstract Context: Sleep loss has immediate effects on metabolic function that in the long run may contribute to the development of obesity and type 2 diabetes. Objective: Our objective was to explore the neuroendocrine mechanisms mediating the acute effects of sleep deprivation on blood glucose regulation under basal and hypoglycemic conditions. Methods: In a randomized, crossover study in 10 healthy young men, plasma concentrations of relevant hormones were examined during basal rest, a subsequent stepwise hypoglycemic clamp after one night of total sleep deprivation (SD) and one night of regular sleep. Results: Basal glucagon concentrations were decreased (P = 0.022) and C-peptide levels were slightly reduced after SD (P = 0.085), compared with regular sleep. During hypoglycemia after SD, the glucagon increase relative to baseline was enhanced (P = 0.034) and the relative decrease in C-peptide was reduced (P = 0.013). Also, the relative increase in norepinephrine was reduced (P = 0.031). SD did not affect epinephrine, ACTH, cortisol, lactate, β-hydroxybutyrate, or nonesterified fatty acids during hypoglycemia, but overall, plasma nonesterified fatty acid levels were reduced after SD (P = 0.009). SD markedly increased rated hunger during basal rest (P < 0.008), resulting in a dampened relative increase during hypoglycemia (P < 0.009). Unexpectedly, despite distinct alterations in basal secretory activity, the absolute amplitude of hormonal counterregulation and hunger responses to hypoglycemia was not affected by SD. Conclusion: Short-term SD distinctly alters hormonal glucose regulation, affecting especially pancreatic islet secretion, and also increases hunger. Immediate perturbations in the dynamic regulation of energy metabolism caused by acute sleep curtailment may contribute to the association between chronic sleep loss and metabolic disorders.