Glucagon plays an important role in glucose homeostasis by regulating hepatic glucose output in both normo- and hypoglycemic conditions. In this study, we created and characterized α cell-specific insulin receptor knockout (αIRKO) mice to directly explore the role of insulin signaling in the regulation of glucagon secretion in vivo. Adult male αIRKO mice exhibited mild glucose intolerance, hyperglycemia, and hyperglucagonemia in the fed state and enhanced glucagon secretion in response to L-arginine stimulation. Hyperinsulinemic-hypoglycemic clamp studies revealed an enhanced glucagon secretory response and an abnormal norepinephrine response to hypoglycemia in αIRKO mice. The mutants also exhibited an age-dependent increase in β cell mass. Furthermore, siRNA-mediated knockdown of insulin receptor in glucagon-secreting InR1G cells promoted enhanced glucagon secretion and complemented our in vivo findings. Together, these data indicate a significant role for intraislet insulin signaling in the regulation of α cell function in both normo- and hypoglycemic conditions.