IGF-I, a known secretory product of intestinal subepithelial myofibroblasts (ISEMFs), is essential for the intestinotropic effects of glucagon-like peptide-2 (GLP-2). Furthermore, GLP-2 increases IGF-I mRNA transcript levels in vitro in heterogeneous fetal rat intestinal cultures, as well as in vivo in the rodent small intestine. To determine the mechanism underlying the stimulatory effect of GLP-2 on intestinal IGF-I mRNA, murine ISEMF cells were placed into primary culture. Immunocytochemistry showed that the ISEMF cells appropriately expressed α-smooth muscle actin and vimentin but not desmin. The cells also expressed GLP-2 receptor and IGF-I mRNA transcripts. Treatment of ISEMF cells with (Gly2)GLP-2 induced IGF-I mRNA transcripts by up to 5-fold of basal levels after treatment with 10(-8) m GLP-2 for 2 h (P < 0.05) but did not increase transcript levels for other intestinal growth factors, such as ErbB family members. Immunoblot revealed a 1.6-fold increase in phospho (p)-Akt/total-(t)Akt with 10(-8) m GLP-2 treatment (P < 0.05) but no changes in cAMP, cAMP-dependent β-galactosidase expression, pcAMP response element-binding protein/tcAMP response element-binding protein, pErk1/2/tErk1/2, or intracellular calcium. Furthermore, pretreatment of ISEMF cells with the phosphatidylinositol 3 kinase (PI3K) inhibitors, LY294002 and wortmannin, abrogated the IGF-I mRNA response to GLP-2, as did overexpression of kinase-dead Akt. The role of PI3K/Akt in GLP-2-induced IGF-I mRNA levels in the murine jejunum was also confirmed in vivo. These findings implicate the PI3K/Akt pathway in the stimulatory effects of GLP-2 to enhance intestinal IGF-I mRNA transcript levels and provide further evidence in support of a role for IGF-I produced by the ISEMF cells in the intestinotropic effects of GLP-2.