The winged helix transcription factors hepatic nuclear factor 3 a, b and g (HNF-3, encoded by the Foxa1, a2 and a3 genes, respectively) are expressed early in embryonic endoderm and play important roles in the regulation of gene expression in liver and pancreas. Foxa1 has been shown to be required for glucagon secretion in the pancreas, while Foxa2 is critical for the regulation of insulin secretion in pancreatic b-cells. Here we address the role of Foxa3 in the maintenance of glucose homeostasis. Mice homozygous for a null mutation in Foxa3 appear normal under fed conditions. However, when fasted, Foxa3-/- mice have a significantly lower blood glucose compared to control mice. The fasting hypoglycemia in Foxa3-/- mice could not be attributed to defects in pancreatic hormone secretion, ketone production or hepatic glycogen breakdown. Surprisingly, mRNA levels for several gluconeogenic enzymes were up-regulated appropriately in fasted Foxa3-/- mice, despite the fact that the corresponding genes had been shown to be activated by Foxa proteins in vitro. However, the mRNA for the plasma membrane glucose transporter GLUT2 was decreased by 64% in the fasted and 93% in the fed state, suggesting that efflux of newly synthesized glucose is limiting in Foxa3-/- hepatocytes. Thus, Foxa3 is the dominating transcriptional regulator of GLUT2 expression in hepatocytes in vivo. In addition, we investigated the hepatic transcription factor network in Foxa3-/- mice and found that the normal activation of HNF 4a and HNF 1a and PGC1 induced by fasting is attenuated in mice lacking Foxa3.