Physiological levels of cortisol have been found to blunt neuroendocrine and metabolic responses to subsequent hypoglycemia in humans. The aim of this study was to determine whether cortisol acts directly on the brain to elicit this effect. A total of 41 conscious unrestrained Sprague-Dawley rats were studied during 2-day experiments. Day 1 consisted of two episodes of clamped 2-h hyperinsulinemic (30 pmol · kg⁻¹ · min⁻¹) hypoglycemia (2.8 ± 0.1 mmol/l; n = 12; ANTE HYPO), euglycemia (6.2 ± 0.1 mmol/l; n = 12; ANTE EUG), or euglycemia (6.2 ± 0.1 mmol/l) plus simultaneous intracerebroventricular (ICV) infusion of cortisol (25 μg/h; n = 9; ANTE EUG+Cort) or saline (24 μl/h; n = 8; ANTE EUG+Sal). For all groups, day 2 consisted of a 2-h hyperinsulinemic (30 pmol · kg⁻¹ · min⁻¹) hypoglycemic (2.9 ± 0.2 mmol/l) clamp. Plasma epinephrine and glucagon incremental area under the curve (ΔAUC) responses were significantly less in ANTE EUG+Cort and ANTE HYPO versus both ANTE EUG and ANTE EUG+Sal (P < 0.05). The ΔAUC responses of plasma norepinephrine were significantly lower in ANTE EUG+Cort versus both ANTE EUG and ANTE EUG+Sal (P < 0.05). Endogenous glucose production was significantly less in ANTE HYPO and ANTE EUG+Cort versus the other groups (P < 0.05). Lastly, the glucose infusion rate to maintain the desired hypoglycemia was significantly greater in ANTE EUG+Cort and ANTE HYPO versus the other two groups (P < 0.05). In summary, ICV infusion of cortisol significantly blunted norepinephrine, epinephrine, glucagon, and endogenous glucose production responses to next-day hypoglycemia. We conclude that cortisol can act directly on the central nervous system to blunt counterregulatory responses to subsequent hypoglycemia in the conscious rat.