Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP/G6PC2) is a major autoantigen in both mouse and human type 1 diabetes. IGRP is selectively expressed in islet beta cells and polymorphisms in the IGRP gene have recently been associated with variations in fasting blood glucose levels and cardiovascular-associated mortality in humans. Chromatin immunoprecipitation (ChIP) assays have shown that the IGRP promoter binds the islet-enriched transcription factors Pax-6 and BETA2. We show here, again using ChIP assays, that the IGRP promoter also binds the islet-enriched transcription factors MafA and Foxa2. Single binding sites for these factors were identified in the proximal IGRP promoter, mutation of which resulted in decreased IGRP fusion gene expression in betaTC-3, Hamster insulinoma tumor (HIT), and Min6 cells. ChiP assays have shown that the islet-enriched transcription factor Pdx-1 also binds the IGRP promoter, but mutational analysis of four Pdx-1 binding sites in the proximal IGRP promoter revealed surprisingly little effect of Pdx-1 binding on IGRP fusion gene expression in betaTC-3 cells. In contrast, in both HIT and Min6 cells mutation of these four Pdx-1 binding sites resulted in a approximately 50% reduction in fusion gene expression. These data suggest that the same group of islet-enriched transcription factors, namely Pdx-1, Pax-6, MafA, BETA2, and Foxa2, directly or indirectly regulate expression of the two major autoantigens in type 1 diabetes.