Glucagon-like peptide-2 (GLP-2) enhances intestinal growth and function through a cAMP-linked G protein-coupled receptor (GPCR) expressed in the mucosal layer and enteric nervous system. Because the type 1B γ-isoform of phosphatidylinositol 3-kinase (PI3-K) is activated by GPCRs, we determined whether this enzyme plays a role in the intestinal actions of GLP-2 by using PI3-Kγ knockout (KO) mice. Wild-type (WT), heterozygous, and KO mice were treated with vehicle or 1 μg Gly2-GLP-2 (a long-acting analog) twice daily for 10 days and analyzed for changes in intestinal growth, motility, and cAMP production. Basal small intestinal wet weight was increased in KO mice in association with enhanced crypt-villus height and crypt cell proliferation ( P < 0.05–0.01). However, the GLP-2-induced changes in these parameters were not different between KO and WT animals. GLP-2 treatment also enhanced the number of mucous cells in the intestinal epithelium, but this effect was lost in the PI3-Kγ KO mice. Both basal and GLP-2-induced suppression of intestinal transit were normal in KO mice. In contrast, the ability of GLP-2 to stimulate cAMP levels in isolated muscle strips was abrogated by loss of PI3-Kγ, despite the expression of GLP-2 receptor mRNA transcripts in this tissue. Together, the results of this study demonstrate a role for PI3-Kγ in basal but not GLP-2-induced small intestinal mucosal growth. However, PI3-Kγ is important for the enhancement of mucous cell number by GLP-2 and in the ability of the GLP-2 receptor to couple to cAMP in the enteric nervous system.