The transcription factor FoxO1 links the phosphatidylinositol 3-kinase (PI 3-kinase) --> Akt cascade to gene expression that regulates cell growth, survival, and metabolism. The receptors for insulin and IGFs factors are linked to this pathway through tyrosine phosphorylation of insulin receptor substrates-Irs1, 2, 3, and 4. However, it is unclear why Irs2 signaling predominates in certain tissues, including pancreatic beta cells, dermal fibroblasts, photoreceptors, central neurons, and metastatic mammary tumor cells. We used wild-type mouse embryo fibroblasts (MEFs)-and Irs1(-/-) or Irs2(-/-) MEFs-to establish the relation between Irs1, Irs2, and FoxO during insulin signaling. PI 3-kinase associated with Irs1 and Irs2 during insulin stimulation of wt MEFs, which strongly promoted Akt and FoxO phosphorylation, led to FoxO nuclear exclusion and degradation. However, insulin failed to activate the Akt--> FoxO cascade in Irs2(-/-) MEFs because Irs1 expression was reduced in these cells, and p110alpha-PI 3-kinase was inefficiently activated during recruitment by Irs1. By contrast, insulin stimulation of Irs1(-/-) MEFs caused FoxO degradation, not only because Irs2 expression increased but also because Irs2 efficiently activated p110alpha--> Akt cascade. Importantly, prolonged insulin stimulation restored FoxO1 expression in wild-type or Irs1(-/-) MEFs because Irs2 was degraded and Irs1 alone failed to activate sufficient p110alpha to promote the Akt--> FoxO cascade. Inhibition of Irs2 degradation with rapamycin caused persistent FoxO degradation even during prolonged insulin stimulation. The dynamic relation between Irs2 and FoxO expression, compared with the subordinate role of Irs1, can explain the dominant role of Irs2 in metabolic regulation.