Glucose-dependent insulinotropic polypeptide (GIP), which is released into the circulation from jejunal mucosal K-cells. Circulating GIP augments pancreatic insulin secretion and hyperinsulinemia plays critical roles in the pathogenesis of obesity and type-2 diabetes mellitus. In recent studies, we have shown GIP directly activates Na-glucose co-transporter-1 (SGLT1) and enhances glucose absorption in mouse jejunum. It is not known whether GIP would also regulates other intestinal nutrient absorptive process(es). Present study investigated the effect of GIP on H⁺-peptide cotransporter-1 (PepT1) that mediates di- and tripeptides as well as peptidomimetic drugs. The activity of PepT1 is driven by a proton (H⁺)-gradient that is, in part, maintained by apical Na:H exchange is form 3 (NHE3). Immunofluorescence studies revealed GIP-receptor and PepT1 like proteins are localized in the villus cells of normal mouse jejunum. Gly-Sar (a relatively non-digestible peptide) fluxes were measured under voltage clamp condition in Ussing type chambers. Mucosal to cytosolic acid pH-gradient (i.e., H⁺ gradient) significantly enhanced the mucosal to serosal Gly-Sar absorption as an evidence for the presence of PepT1 (i.e., H⁺-Gly-Sar cotransports) on the apical membranes of mouse jejunum. The H⁺-gradient driven Gly-Sar absorption was: 1) completely inhibited by cephalexin (a competitive inhibitor of Pept1); 2 significantly activated by GIP; and 3) not affected by GLP-1. The GIP-activated Gly-Sar absorption was completely inhibited by RP-cAMP (a cAMP-antagonist). These observations indicate that GIP directly activates the Pept1 activity by a cAMP-dependent signaling pathway in jejunum.