Glucose-dependent insulinotropic polypeptide (GIP) potentiates glucose-stimulated insulin secretion (GSIS). This response is blunted in type 2 diabetes (T2DM). Xenin-25 is a 25-amino acid neurotensin-related peptide that amplifies GIP-mediated GSIS in hyperglycemic mice. This study determines if xenin-25 amplifies GIP-mediated GSIS in humans with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), or T2DM. Each fasting subject received graded glucose infusions to progressively raise plasma glucose concentrations, along with vehicle alone, GIP, xenin-25, or GIP plus xenin-25. Plasma glucose, insulin, C-peptide, and glucagon levels and insulin secretion rates (ISRs) were determined. GIP amplified GSIS in all groups. Initially, this response was rapid, profound, transient, and essentially glucose independent. Thereafter, ISRs increased as a function of plasma glucose. Although magnitudes of insulin secretory responses to GIP were similar in all groups, ISRs were not restored to normal in subjects with IGT and T2DM. Xenin-25 alone had no effect on ISRs or plasma glucagon levels, but the combination of GIP plus xenin-25 transiently increased ISR and plasma glucagon levels in subjects with NGT and IGT but not T2DM. Since xenin-25 signaling to islets is mediated by a cholinergic relay, impaired islet responses in T2DM may reflect defective neuronal, rather than GIP, signaling.