Not only in the central nervous system, γ-aminobutyric acid (GABA) is also produced in and released from pancreatic β-cells. However, physiological roles of pancreatic GABA are not fully understood. GABA is produced by two isoforms of glutamate decarboxylase GAD67 and GAD65, which are encoded by the Gad1 and Gad2 genes, respectively. While GABAergic neurons express both GAD67 and GAD65, pancreatic β-cells only express GAD67 in mice. In this study, we developed the mice lacking GABA production in pancreatic β-cells by deleting the Gad1 gene from these cells, and examined the glucose tolerance in those mice. The GABA-immunoreactivity (ir) was detected in the wild-type mouse pancreatic islets. In contrast, the GABA-ir was completely eliminated in Gad1^flox/flox;Rip-Cre mice (Gad1-βKO mice), indicating the Gad1 gene deletion from β-cells in the mice. The blood glucose levels under baseline and fasting condition were comparable between untreated Gad1-βKO mice and the control mice. However, under the fasting condition, the blood glucose levels were significantly lower in Gad1-βKO mice than in the control mice 30 min after the treatment with glucose (2 g/kg, p.o.). These results indicate that the loss of GABA production in pancreatic β-cells may promote insulin secretion and suppress the elevation in blood glucose levels.