Transcriptional regulation of insulin in pancreatic β-cells is mediated primarily through enhancer elements located within the 5' upstream regulatory region of the preproinsulin gene. Recently, the Krüppel-like transcription factor, Gli-similar 3 (Glis3), was shown to bind the insulin (INS) promoter and positively influence insulin transcription. In this report, we examined in detail the synergistic activation of insulin transcription by Glis3 with coregulators, CREB-binding protein (CBP)/p300, pancreatic and duodenal homeobox 1 (Pdx1), neuronal differentiation 1 (NeuroD1), and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA). Our data show that Glis3 expression, the binding of Glis3 to GlisBS, and its recruitment of CBP are required for optimal activation of the insulin promoter in pancreatic β-cells not only by Glis3, but also by Pdx1, MafA, and NeuroD1. Mutations in the GlisBS or small interfering RNA-directed knockdown of GLIS3 diminished insulin promoter activation by Pdx1, NeuroD1, and MafA, and neither Pdx1 nor MafA was able to stably associate with the insulin promoter when the GlisBS were mutated. In addition, a GlisBS mutation in the INS promoter implicated in the development of neonatal diabetes similarly abated activation by Pdx1, NeuroD1, and MafA that could be reversed by increased expression of exogenous Glis3. We therefore propose that recruitment of CBP/p300 by Glis3 provides a scaffold for the formation of a larger transcriptional regulatory complex that stabilizes the binding of Pdx1, NeuroD1, and MafA complexes to their respective binding sites within the insulin promoter. Taken together, these results indicate that Glis3 plays a pivotal role in the transcriptional regulation of insulin and may serve as an important therapeutic target for the treatment of diabetes.