Forkhead box O (FOXO) transcription factors have been implicated in regulating the metabolism, cellular proliferation, stress resistance, apoptosis, and longevity. Through the insulin receptor substrate → phosphoinositide 3-kinase → Akt signal cascade, FOXO integrates insulin action with the systemic nutrient and energy homeostasis. Activation of FOXO1 in liver induces gluconeogenesis via phosphoenolpyruvate carboxykinase (PEPCK)/glucose 6-phosphate pathway, and disrupts mitochondrial metabolism and lipid metabolism via heme oxygenase 1/sirtuin 1/Ppargc1α pathway. In skeletal muscle, FOXO1 activation underpins the carbohydrate/lipid switch during fasting state. Inhibition of FOXO1 under physiological conditions accounts for maintenance of skeletal muscle mass/function and adipose differentiation. In pancreatic β-cells, nuclear translocation of FOXO1 antagonizes pancreatic and duodenal homeobox 1 and attenuates β-cells proliferation and insulin secretion. Regardless, FOXO1 promotes the proliferation of β-cells through induction of Cyclin D1 in low nutrition, and elicits antioxidant mechanism to protect against β-cell failure during oxidative insults. In the brain, FOXO1 controls food intake through transcriptional regulation of the orexigenic neuropeptide Y, agouti-related protein, and carboxypeptidase E. In this article, we review the role of FOXO1 in the regulation of metabolism and energy expenditure based on recent findings from mouse models, and discuss the therapeutic value of targeting FOXO1 in metabolic diseases. Antioxid. Redox Signal. 14, 649–661.