Our previous studies on cardiac myocytes showed that positive inotropic concentrations of the digitalis drug ouabain activated signaling pathways linked to Na⁺/K⁺-ATPase through Src and EGFR, and led to myocyte hypertrophy. In view of the known involvement of PI3K/Akt pathways in cardiac hypertrophy, the aim of this work was to determine if these pathways are also linked to cardiac Na⁺/K⁺-ATPase, and if so, to assess their role in ouabain-induced myocyte growth. In a dose- and time-dependent manner, ouabain activated Akt, and its substrates m-TOR and GSK, in neonatal rat cardiac myocytes. Akt activation by ouabain was sensitive to PI3K inhibitors, and was also noted in adult myocytes and isolated hearts. Ouabain caused transient increase of PIP3 content of neonatal myocytes, activated class IA, but not class IB, PI3K, and increased coimmunoprecipitation of -subunit of Na⁺/K⁺-ATPase with p85 subunit of class IA PI3K. Ouabain-induced activation of ERK1/2 was prevented by inhibitors of Src, EGFR, and MEK, but not by PI3K inhibitors. Activation of Akt by ouabain, however, was sensitive to inhibitors of PI3K and Src, but not to those of EGFR and MEK. Similarly, ouabain-induced myocyte hypertrophy was prevented by PI3K and Src inhibitors, but not by an EGFR inhibitor. These findings (a) establish the linkage of class IA PI3K/Akt pathway to Na⁺/K⁺-ATPase, and the essential role of this linkage to ouabain-induced myocyte hypertrophy; and (b) suggest cross-talk between these PI3K/Akt pathways and the signaling cascades previously identified to be associated with cardiac Na⁺/K⁺-ATPase.