The nuclear receptors CAR and PXR activate hepatic genes in response to therapeutic drugs and xenobiotics, leading to the induction of drug-metabolizing enzymes, such as cytochrome P450. Insulin inhibits the ability of FOXO1 to express genes encoding gluconeogenic enzymes. Induction by drugs is known to be decreased by insulin, whereas gluconeogenic activity is often repressed by treatment with certain drugs, such as phenobarbital (PB). Performing cell-based transfection assays with drug-responsive and insulin-responsive enhancers, glutathione S-transferase pull down, RNA interference (RNAi), and mouse primary hepatocytes, we examined the molecular mechanism by which nuclear receptors and FOXO1 could coordinately regulate both enzyme pathways. FOXO1 was found to be a coactivator to CAR- and PXR-mediated transcription. In contrast, CAR and PXR, acting as corepressors, downregulated FOXO1-mediated transcription in the presence of their activators, such as 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and pregnenolone 16alpha-carbonitrile, respectively. A constitutively active mutant of the insulin-responsive protein kinase Akt, but not the kinase-negative mutant, effectively blocked FOXO1 activity in cell-based assays. Thus, insulin could repress the receptors by activating the Akt-FOXO1 signal, whereas drugs could interfere with FOXO1-mediated transcription by activating CAR and/or PXR. Treatment with TCPOBOP or PB decreased the levels of phosphoenolpyruvate carboxykinase 1 mRNA in mice but not in Car(-/-) mice. We conclude that FOXO1 and the nuclear receptors reciprocally coregulate their target genes, modulating both drug metabolism and gluconeogenesis.