To clarify the role of insulin in modulating the glucagon response to glucose concentration changes, we investigated the effects of exogenous insulin (10 mU/mL, 100 mU/mL, and 3.3 U/mL) on responses to high glucose (5.6 → 16.7 mmol/L), low glucose (5.6 → 1.4 mmol/L), and arginine (10 mmol/L) stimulation using the perfused rat pancreas. Although glucagon levels were slightly suppressed by all of the exogenous insulin concentrations tested for the initial few minutes at 5.6 mmol/L glucose, baseline levels were maintained thereafter. Glucagon responses to high or normal glucose concentrations were not altered, but glucopenia-induced glucagon secretion was significantly suppressed as compared with that of controls (0.77 ± 0.14 ng/min [10 mU/mL, n = 5], 0.55 ± 0.14 ng/min [100 mU/mL, n = 5], 0.27 ± 0.13 ng/min [3.3 U/mL, n = 5] v 1.38 ± 0.20 ng/min [controls, n = 9], P < .05, respectively). The first phase of the glucagon response to arginine was potentiated (2.03 ± 0.24 v 1.17 ± 0.22 ng/min, P < .05) by 10 mU/mL exogenous insulin. The second phase of the glucagon response to arginine was significantly suppressed in the presence of higher concentrations of exogenous insulin (1.16 ± 0.23 ng/min [100 mU/mL], 0.96 ± 0.08 ng/min [3.3 U/mL] v 1.57 ± 0.17 ng/min, P < .05, respectively). These results suggest that glucagon secretion is modified by the combined suppressive effects of glucose and insulin, although it is mainly glucose that mediates glucagon secretion in the physiological glucose range. Glucopenia- or arginine-induced glucagon secretion is suppressed by insulin.