CONTEXT: Insulin action in the human brain influences eating behavior, cognition, and whole-body metabolism. Studies investigating brain insulin rely on intranasal application. OBJECTIVE: To investigate effects of three doses of insulin and placebo as nasal sprays on the central and autonomous nervous system and analyze absorption of insulin into the bloodstream. DESIGN, PARTICIPANTS, AND METHODS: Nine healthy men received placebo or 40 U, 80 U, and 160 U insulin spray in randomized order. Before and after spray, brain activity was assessed by functional magnetic resonance imaging, and heart rate variability (HRV) was assessed from electrocardiogram. Plasma insulin, C-peptide, and glucose were measured regularly. SETTING: General community. RESULTS: Nasal insulin administration dose-dependently modulated regional brain activity and the normalized high-frequency component of the HRV. Post hoc analyses revealed that only 160 U insulin showed a considerable difference from placebo. Dose-dependent spillover of nasal insulin into the bloodstream was detected. The brain response was not correlated with this temporary rise in circulating insulin. CONCLUSIONS: Nasal insulin dose-dependently modulated regional brain activity with the strongest effects after 160 U. However, this dose was accompanied by a transient increase in circulating insulin concentrations due to a spillover into circulation. Our current results may serve as a basis for future studies with nasal insulin to untangle brain insulin effects in health and disease.