The regulation of Na/K-ATPase in various tissues is under the control of a number of hormones and peptides that exert both short- and long-term control over its activity. Present study was performed to investigate the effect of chronic insulin treatment on Na/K-ATPase in renal proximal tubular cells. Incubation of opossum kidney (OK) cells, transfected with rat Na/K-ATPase α1- subunit, with 1 nmol/L insulin for 48hr decreased Na/K-ATPase activity. Insulin decreased α1 protein content and increased α1 serine phosphorylation and α1-adaptor protein (AP) 2 interaction. Removal of 26 N-terminal (-NT) amino acid from α1-subunit containing serine/threonine sites abolished the insulin-mediated serine-phosphorylation and inhibition of Na/K-ATPase. Substitution of serine 16 and 23 with alanine showed comparable effect to -NT. Insulin increased the activity of protein kinase (PK) C which was blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. Both PI3K and PKC inhibitors abolished the insulin mediated inhibition of Na/K-ATPase. Insulin increased the expression of PKC β1, , and , however, only PKC / specific inhibitors blocked insulin-induced phosphorylation and inhibition of Na/K-ATPase. Our data demonstrate that insulin activates atypical PKC isoforms / via PI3K pathway. PKC /-induced phosphorylation of α1-subunit at serine 16 and 23 leads to AP-2 recruitment, degradation and a decrease in Na/K-ATPase activity.