An aberrant increase in circulating catabolic hormone glucagon contributes to type 2 diabetes pathogenesis. However, mechanisms regulating glucagon secretion and α-cell mass are not well understood. In this study, we aimed to demonstrate that phosphatidylinositol 3-kinase (PI3K) signaling is an important regulator of α-cell function. Mice with deletion of PTEN, a negative regulator of this pathway, in α-cells show reduced circulating glucagon levels and attenuated L-arginine–stimulated glucagon secretion both in vivo and in vitro. This hypoglucagonemic state is maintained after high-fat–diet feeding, leading to reduced expression of hepatic glycogenolytic and gluconeogenic genes. These beneficial effects protected high-fat diet–fed mice against hyperglycemia and insulin resistance. The data demonstrate an inhibitory role of PI3K signaling on α-cell function and provide experimental evidence for enhancing α-cell PI3K signaling for diabetes treatment.