Aims: Incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gastrointestinal hormones that are released in response to nutrient intake and promote insulin secretion via G-protein coupled receptors. Interestingly, a subset of enteroendocrine cells expresses both GIP and GLP-1. Proglucagon is differently processed in the gut and the pancreas. We sought to determine whether GIP also might be expressed as an isoform and co-expressed with proglucagon in pancreatic α-cells. Methods: We assessed GIP expression via RT-PCR, in situ hybridization, and immunohistochemistry. We measured GIP release from isolated islets via the bioassay, compared the biological activities of full-length and short-from GIP, and assessed the impact of immunoneutralization of islet GIP on glucose-stimulated insulin secretion in isolated islets. We also investigated whether short-from GIP is expressed in the gut. Results: GIP mRNA was detected in mouse islets. GIP protein localized to pancreatic α-cells of mouse, human, and snake, based on immunohistochemical analyses. However, immunoreactivity was detected in islets from pro-hormone convertase (PC) 2 knockout but not wild-type mice by a C-terminal GIP antibody. Intriguingly, we found short-form GIP expression with PC2 especially in the lower gut. Bioactive GIP was released from mouse and human islets after arginine stimulation. In several diabetic animal models, GIP expression is concomitant with α-cells expansion in the islets. In the perfused mouse pancreas, GIP1-42 and GIP1-30 had equipotent insulinotropic actions. Finally, immunoneutralization of GIP secreted by isolated islets decreased glucose-stimulated insulin secretion. Conclusions: GIP is expressed in and released from pancreatic islets; in α-cells, PC2 processes proGIP to a short-form but bioactive form of GIP that differs from the full-length, PC1/3-derived GIP form from K-cells. Short-form GIP is also expressed in the gut. Islet-derived GIP promotes islet glucose competence and also could support islet development and survival.