Endogenous cardiotonic glycosides bind to the inhibitory binding site of the plasma membrane sodium pump (Na⁺/K⁺ ATPase). Plasma levels of endogenous cardiotonic glycosides increase in several disease states like essential hypertension and uremia. Low concentrations of ouabain, which do not inhibit Na⁺/K⁺ ATPase, induce cell proliferation. The mechanisms of ouabain-mediated response remain unclear. Recently, we demonstrated that in opossum kidney (OK) proximal tubular cells, low concentrations of ouabain induce cell proliferation through phosphorylation of protein kinase B (Akt) in a calcium dependent manner. In the present study, we identified ERK as an upstream kinase regulating Akt activation in ouabain-stimulated cells. Further, we provide evidence that low concentrations of ouabain stimulate Na⁺/K⁺ ATPase-mediated ⁸⁶Rb uptake in an Akt, ERK, and Src kinase-dependent manner. Ouabain-mediated ERK phosphorylation was inhibited by blockade of intracellular calcium release, calcium entry, tyrosine kinases, and phospholipase C. Inhibition of PI-3K and Akt failed to inhibit ouabain-stimulated ERK phosphorylation. Ouabain-mediated Akt phosphorylation was inhibited by U0126, a MEK/ERK inhibitor, suggesting that ouabain-mediated Akt phosphorylation is dependent on ERK. In an in vitro kinase assay, active recombinant ERK phosphorylated recombinant Akt on Ser⁴⁷³. Moreover, transient transfection with constitutively active MEK1, an upstream regulator of ERK, increased Akt phosphorylation and activation, while over-expression of constitutively active Akt, failed to stimulate ERK phosphorylation. Ouabain at low concentrations also promoted cell proliferation in an ERK-dependent manner. These findings suggest that ouabain-stimulated ERK phosphorylation is required for Akt phosphorylation on Ser⁴⁷³, cell proliferation, and stimulation of Na⁺/K⁺ ATPase-mediated 86Rb uptake in OK cells.