Cardiomyocyte apoptosis contributes toward the loss of muscle mass in myocardial pathologies. Previous reports have implicated type I cAMP-dependent protein kinase (PKA) and p90 ribosomal S6 kinase (RSK) in cardiomyocyte apoptosis. However, the precise mechanisms and the isoform of RSK involved in this process remain undefined. Using adult rat ventricular myocytes and mouse-derived cardiac HL-1 cardiomyocytes, we demonstrate that hypoxia/reoxygenation (H/R)-induced apoptosis is accompanied by a decrease in the type I PKA regulatory subunit (PKARIα) and activation of RSK1. As previously described by us for other cell types, in cardiomyocytes, inactive RSK1 also interacts with PKARIα, whereas the active RSK1 interacts with the catalytic subunit of PKA. Additionally, small interfering (siRNA)-mediated silencing of PKARIα or disrupting the RSK1/PKARIα interactions with a small, cell-permeable peptide activates RSK1 and recapitulates the H/R-induced apoptosis. Inhibition of RSK1 or siRNA-mediated silencing of RSK1 attenuates H/R-induced apoptosis, demonstrating the role of RSK1 in cardiomyocyte apoptosis. Furthermore, silencing of RSK1 decreases the H/R-induced phosphorylation of sodium–hydrogen exchanger 1 (NHE1), and inhibition of NHE1 with 5′-N-ethyl-N-isopropyl-amiloride blocks H/R induced apoptosis, indicating the involvement of NHE1 in apoptosis. Overall, our findings demonstrate that H/R-mediated decrease in PKARIα protein levels leads to activation of RSK1, which via phosphorylation of NHE1 induces cardiomyocyte apoptosis.