The epithelial sodium channel (ENaC) plays an important role in transepithelial Na(+) absorption; hence its function is essential for maintaining Na(+) and fluid homeostasis and regulating blood pressure. Insulin is one of the hormones that regulates activity of ENaC. In this study, we investigated the contribution of two related protein kinases, Akt (also known as protein kinase B) and the serum- and glucocorticoid-dependent kinase (Sgk), on insulin-induced ENaC activity in Fisher rat thyroid cells expressing ENaC. Overexpression of Akt1 or Sgk1 significantly increased ENaC activity, whereas expression of a dominant-negative construct of Akt1, Akt1(K179M), decreased basal activity of ENaC. Inhibition of the endogenous expression of Akt1 and Sgk1 by short interfering RNA not only inhibited ENaC but also disrupted the stimulatory effect on ENaC of insulin and of the downstream effectors of insulin, phosphatidylinositol 3-kinase and PDK1. Conversely, overexpression of Akt1 or Sgk1 increased expression of ENaC at the cell membrane and overcame the inhibitory effect of Nedd4-2 on ENaC. Furthermore, mutation of consensus phosphorylation sites on Nedd4-2 for Akt1 and Sgk1, Ser(342) and Ser(428), completely abolished the inhibitory effect of Sgk1 and Akt1 on Nedd4-2 action. Together these data suggest that both Akt and Sgk are components of an insulin signaling pathway that increases Na(+) absorption by up-regulating membrane expression of ENaC via a regulatory system that involves inhibition of Nedd4-2.