Glucagon-like peptide-1 (GLP-1) increases β-cell function and growth through protein kinase A- and phosphatidylinositol-3-kinase (PI3-K)/protein kinase B, respectively. GLP-1 acts via a G protein-coupled receptor, and PI3-Kγ is known to be activated by Gβγ. Therefore, the role of PI3-Kγ in the chronic effects of GLP-1 on the β-cell was investigated using PI3-Kγ knockout (KO) mice treated with the GLP-1 receptor agonist, exendin-4 (Ex4; 1 nmol/kg sc every 24 h for 14 d). In vivo, glucose and insulin responses were similar in PBS- and Ex4-treated KO and wild-type (WT) mice. However, glucose-stimulated insulin secretion was markedly impaired in islets from PBS-KO mice (P < 0.05), and this was partially normalized by chronic Ex4 treatment (P < 0.05). In contrast, insulin content was increased in PBS-KO islets, and this was paradoxically decreased by Ex4 treatment, compared with the stimulatory effect of Ex4 on WT islets (P < 0.05–0.01). Transfection of INS-1E β-cells with small interfering RNA for PI3-Kγ similarly decreased glucose-stimulated insulin secretion (P < 0.01) and increased insulin content. Basal values for β-cell mass, islet number and proliferation, glucose transporter 2, glucokinase, and insulin receptor substrate-2 were increased in PBS-KO mice (P < 0.05–0.001) and, although they were increased by Ex4 treatment of WT animals (P < 0.05), they were decreased in Ex4-KO mice (P < 0.05–0.01). These findings indicate that PI3-Kγ deficiency impairs insulin secretion, resulting in compensatory islet growth to maintain normoglycemia. Chronic Ex4 treatment normalizes the secretory defect, thereby relieving the pressure for expansion of β-cell mass. These studies reveal a new role for PI3-Kγ as a positive regulator of insulin secretion, and reinforce the importance of GLP-1 for the maintenance of normal β-cell function.