The Forkhead box A2 transcription factor (Foxa2/HNF-3beta) has been shown to be a key regulator of genes involved in the maintenance of glucose and lipid homeostasis in the liver. It is constitutively inactivated in several hyperinsulinemic/obese mouse models, thereby enhancing their metabolic phenotypes. Foxa2 is activated under fasting conditions but is inhibited by insulin signaling via phosphatidylinositol 3-kinase/AKT in a phosphorylation-dependent manner, which results in its nuclear exclusion. However, the mechanism and relative importance of its nuclear export has not yet been elucidated. Here we show that Foxa2 contains a functional nuclear export signal and is excluded from the nucleus via a CRM1-dependent pathway in response to insulin signaling. Furthermore, direct evidence is provided that nuclear export-defective Foxa2 is phosphorylated and inactivated by insulin in vitro and in vivo. These data demonstrate for the first time that phosphorylation itself is the main event regulating the activity of Foxa2, suggesting that export-independent mechanisms have evolved to ensure inhibition of Foxa2 under conditions in which insulin signaling is present.