Decreases in cardiac Na/K-ATPase have been documented in patients with heart failure. Reduction of Na/K-ATPase α1 also contributes to the deficiency in cardiac contractility in animal models. Our previous studies demonstrate that reduction of cellular Na/K-ATPase causes cell growth inhibition and cell death in renal proximal tubule cells. To test whether reduction of Na/K-ATPase in combination with increased cardiotonic steroids causes cardiac myocyte death and cardiac dysfunction, we examined heart function in Na/K-ATPase α1 heterozygote knock-out mice (α1(+/-)) in comparison to wild type (WT) littermates after infusion of marinobufagenin (MBG). Adult cardiac myocytes were also isolated from both WT and α1(+/-) mice for in vitro experiments. The results demonstrated that MBG infusion increased myocyte apoptosis and induced significant left ventricle dilation in α1(+/-) mice but not in their WT littermates. Mechanistically, it was found that in WT myocytes MBG activated the Src/Akt/mTOR signaling pathway, which further increased phosphorylation of ribosome S6 kinase (S6K) and BAD (Bcl-2-associated death promoter) and protected cells from apoptosis. In α1(+/-) myocytes, the basal level of phospho-BAD is higher compared with WT myocytes, but MBG failed to induce further activation of the mTOR pathway. Reduction of Na/K-ATPase also caused the activation of caspase 9 but not caspase 8 in these cells. Using cultures of neonatal cardiac myocytes, we demonstrated that inhibition of the mTOR pathway by rapamycin also enabled MBG to activate caspase 9 and induce myocyte apoptosis.