Heterozygosity for the Pax6 allele is associated with impaired glucose tolerance in humans. With a Pax6 mutant mice model, we found many of the metabolic abnormalities were consistent with the effects of down-regulating the expression of glucagon-like peptide 1 (GLP-1). In addition to impaired glucose tolerance, adult heterozygous mutant mice (Pax6^m/+) secreted less insulin responding to glucose and arginine administration compared to control mice. Moreover, Pax6^m/+ mice showed increased food intake than control mice although they were resistant to diet-induced fat accumulation. Indeed levels of circulating GLP-1 and intestinal transcription of Gcg/Proglucagon were dramatically reduced in Pax6^m/+ mice. Mutated Pax6 also failed to activate the Gcg/Proglucagon promoter by in vitro transfection assay. Finally, administering the GLP-1 receptor agonist exendin-4 to Pax6^m/+ mice largely reversed their abnormal food intake, glycemic excursion, and insulin secretion. Our studies suggested that disruption of metabolic homeostasis mainly caused by Pax6 haploinsufficiency was mainly mediated by down-regulation of GLP-1. Administration of exendin-4 may be a useful therapy in humans with similar mutation.