MafA is a transcription factor that binds to the promoter in the insulin gene and has been postulated to regulate insulin transcription in response to serum glucose levels, but there is no current in vivo evidence to support this hypothesis. To analyze the role of MafA in insulin transcription and glucose homeostasis in vivo, we generated MafA-deficient mice. Here we report that MafA mutant mice display intolerance to glucose and develop diabetes mellitus. Detailed analyses revealed that glucose-, arginine-, or KCl-stimulated insulin secretion from pancreatic beta cells is severely impaired, although insulin content per se is not significantly affected. MafA-deficient mice also display age-dependent pancreatic islet abnormalities. Further analysis revealed that insulin 1, insulin 2, Pdx1, Beta2, and Glut-2 transcripts are diminished in MafA-deficient mice. These results show that MafA is a key regulator of glucose-stimulated insulin secretion in vivo.