Foxa1 and Foxa2 play both redundant and distinct roles in early pancreas development. We demonstrate here that inducible ablation of both transcription factors in mature mouse β-cells leads to impaired glucose homeostasis and insulin secretion. The defects in both glucose-stimulated insulin secretion and intracellular calcium oscillation are more pronounced than those in β-cells lacking only Foxa2. Unexpectedly, in contrast to the severe reduction of β-cell-enriched factors contributing to metabolic and secretory pathways, expression of a large number of genes that are involved in neural differentiation and function is significantly elevated. We further demonstrate that expression of carbohydrate response element-binding protein (ChREBP or Mlxipl), an important transcriptional regulator of carbohydrate metabolism, is significantly affected in compound Foxa1/a2 mutant β-cells. ChREBP expression is directly controlled by Foxa1 and Foxa2 in both the fetal endocrine pancreas as well as mature islets. These data demonstrate that Foxa1 and Foxa2 play crucial roles in the development and maintenance of β-cell-specific secretory and metabolic pathways.