Antecedent hypoglycemia blunts counterregulatory responses that normally restore glycemia, a phenomenon known as hypoglycemia-associated autonomic failure (HAAF). The mechanisms leading to impaired counterregulatory responses are largely unknown. Hypothalamic AMPK acts as a glucose sensor. To determine whether failure to activate AMPK could be involved in the etiology of HAAF, we developed a model of HAAF using repetitive intracerebroventricular (ICV) injection of 2-deoxy-D-glucose (2DG) resulting in transient neuroglucopenia in normal rats. Ten minutes after a single ICV injection of 2DG, both 1- and 2-AMPK activities were increased 30-50% in arcuate and ventromedial/dorsomedial hypothalamus but not in other hypothalamic regions, hindbrain or cortex. Increased AMPK activity persisted in arcuate at 60 min after 2DG injection when serum glucagon and corticosterone levels were increased 2.5-3.4 fold. When 2DG was injected ICV daily for 4 days, hypothalamic 1- and 2-AMPK responses were markedly blunted in arcuate, and 1-AMPK was also blunted in MH, 10 min after 2DG on day 4. Both AMPK isoforms were activated normally in arcuate at 60 min. Counterregulatory hormone responses were impaired by recurrent neuroglucopenia and were partially restored by ICV injection of, 5-aminoimidazole-4-carboxamide, an AMPK activator, prior to 2DG. Glycogen content increased 2-fold in hypothalamus after recurrent neuroglucopenia, suggesting that glycogen supercompensation could be involved in down-regulating the AMPK glucose-sensing pathway in HAAF. Thus, activation of hypothalamic AMPK may be important for the full counterregulatory hormone response to neuroglucopenia. Furthermore, impaired or delayed AMPK activation in specific hypothalamic regions may play a critical role in the etiology of HAAF.