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. As the type 1B -isoform of PI3-K is activated by GPCRs, we determined whether this enzyme plays a role in the intestinal actions of GLP-2, using PI3-K knock-out (KO) mice. Wild-type (WT), heterozygous and KO mice were treated with vehicle or 1 µg/d 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-K 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-K, despite the expression of GLP-2 receptor mRNA transcripts in this tissue. Together, the results of this study demonstrate a role for PI3-K in basal but not GLP-2-induced small intestinal mucosal growth. However, PI3-K 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.