To determine the role of the autonomic nervous system (ANS) in mediating the glucagon response to marked insulin-induced hypoglycemia in dogs, we measured arterial and pancreatic venous glucagon responses to insulin-induced hypoglycemia during acute, terminal experiments in halothane-anesthetized dogs in which the ANS was intact (control; n = 9), pharmacologically blocked by the nicotinic ganglionic antagonist hexamethonium (n = 6), or surgically ablated by cervical vagotomy and cervical spinal cord section (n = 6). In control dogs, insulin injection caused plasma glucose to fall by 4.4 ± 0.2 mM to a nadir of 1.7 ± 0.2 mM. Arterial epinephrine (EPI) levels increased by 13,980 ± 1860 pM (P < 0.005), confirming marked activation of the ANS. Pancreatic output of glucagon increased from 0.53 ± 0.12 to 2.04 ± 0.38 ng/min during hypoglycemia (change [Δ] + 1.51 ± 0.33 ng/min, P < 0.005). This increased arterial plasma glucagon from 27 ± 3 to 80 ± 15 ng/L (Δ + 52 ± 14 ng/L, P < 0.025). Hexamethonium markedly reduced the ANS response to insulin injection (ΔEPI + 2130 ± 600 pM, P < 0.025 vs. control) despite a similar fall of plasma glucose (Δ −4.1 ± 0.2 mM) and a lower nadir (0.6 ± 0.1 mM). Both the pancreatic glucagon response (delta glucagon output +0.45 ± 0.2 ng/min) and the arterial immunoreactive glucagon response (Δ + 5 ± 4 ng/L) were substantially reduced by hexamethonium (P < 0.025). Vagotomy plus spinal cord section totally abolished the arterial EPI response to insulin injection despite a larger fall of plasma glucose (Δ −5.4 ± 0.4 mM) and a lower nadir (0.9 ± 0.2 mM). Again, both the pancreatic glucagon response (Δglucagon output +0.27 ± 0.23 ng min) and the arterial glucagon response (Δ +5 ± 2 ng/L) were significantly reduced (both P < 0.025 vs. control). We conclude that autonomic activation contributes to the glucagon response to marked insulin-induced hypoglycemia in dogs.