Regulation of insulin gene expression in response to increases in blood glucose levels is essential for maintaining normal glucose homeostasis; however, the exact mechanisms by which glucose stimulates insulin gene transcription are not known. We have shown previously that glucose stimulates insulin gene expression by causing the hyperacetylation of histone H4 at the insulin promoter. We demonstrate that the histone acetyltransferase p300 is recruited to the insulin promoter only at high concentrations of glucose via its interaction with the β-cell-specific transcription factor Pdx-1. Disruption of the function of the endogenous Pdx-1 abolishes the recruitment of p300 to the insulin gene promoter at high concentrations of glucose and results in decreased histone H4 acetylation and insulin gene expression. Furthermore, we demonstrate that the glucose-dependent interaction of Pdx-1 with p300 is regulated by a phosphorylation event that changes the localization of Pdx-1. Based on these data, we conclude that hyperacetylation of histone H4 at the insulin gene promoter in response to high concentrations of glucose depends on the β-cell-specific transcription factor Pdx-1, which is required for the recruitment of the histone acetyltransferase p300 to the insulin gene promoter.