OBJECTIVE Hypoglycemia, the limiting factor in the glycemic management of diabetes, is the result of the interplay of therapeutic insulin excess and compromised glycemic defenses. The key feature of the latter is an attenuated sympathoadrenal response to hypoglycemia that typically follows an episode of recent antecedent iatrogenic hypoglycemia, a phenomenon termed hypoglycemia-associated autonomic failure (HAAF) in diabetes. We investigated the role of cerebral mechanisms in HAAF by measuring regional brain activation during recurrent hypoglycemia with attenuated counterregulatory responses and comparing it with initial hypoglycemia in healthy individuals. METHODS We used [(15)O]water and positron emission tomography to measure regional cerebral blood flow as a marker of brain synaptic activity during hyperinsulinemic hypoglycemic clamps (55 mg/dl [3.0 mmol/l]) in the naïve condition (day 1) and after approximately 24 h of interval interprandial hypoglycemia (day 2) in nine healthy adults. RESULTS Interval hypoglycemia produced attenuated sympathoadrenal, symptomatic, and other counterregulatory responses to hypoglycemia on day 2, a model of HAAF. Synaptic activity in the dorsal midline thalamus during hypoglycemia was significantly greater on day 2 than day 1 (P = 0.004). CONCLUSIONS Greater synaptic activity associated with attenuated counterregulatory responses indicates that the dorsal midline thalamus plays an active inhibitory role in reducing sympathoadrenal and symptomatic responses to hypoglycemia when previous hypoglycemia has occurred, the key feature of HAAF in diabetes.