A gender difference in the glucagon response to insulin-induced hypoglycemia was previously demonstrated in humans. Whether this reflects a gender difference in autonomic activation or in pancreatic -cell regulation is not known. We investigated the glucagon, epinephrine, and norepinephrine responses to neuroglycopenic stress induced by 2-deoxy-D-glucose (2-DG) or insulin in female and male mice. 2-DG increased plasma glucagon levels by 559 ± 68% in females versus 281 ± 46% in males (P < 0.01). Plasma levels of epinephrine or norepinephrine after 2-DG administration did not differ between genders. During insulin-induced hypoglycemia, the glucagon response was similarly higher in females (P < 0.001), whereas the plasma catecholamine response was higher in males (P < 0.05). In vivo, the glucagon response to carbachol or clonidine was higher in females (P < 0.05). In isolated islets, the glucagon response to carbachol (100 µM; P = 0.003) but not to clonidine (1 µM) was larger in females. We conclude that in addition to a larger -cell mass (previously described in female mice), an increased sensitivity of the glucagon-producing -cell to cholinergic activation contributes to the larger glucagon response to glucopenic stress in female mice.