Recently we have reported potent anti-cancer actions of various steroidal Na+/K+ ATPase inhibitors in multiple cell lines. Furthermore, the most powerful compound identified in this study, the 3-[(R)-3-pyrrolidinyl]oxime derivative (3-R-POD), was highly effective in various tumor cell lines in vitro, and exhibited significant tumor growth inhibition in prostate and lung xenografts in vivo. In the present study we have addressed the molecular mechanisms implicated in the anti-cancer actions of 3-R-POD. We report here that 3-R-POD induces strong apoptotic responses in A549 lung- and in DU145 prostate- cancer cells. These effects are accompanied by significant upregulation of caspase-3 activity. Focussing on A549 cells, we further demonstrate late downregulation of BCL-2- and upregulation of c-Fos- gene transcription. In addition, the steroidal Na+/K+ ATPase inhibitor induced late dephosphorylation of Focal Adhesion Kinase (FAK) and activation of p38 MAPK. Our findings suggest that the steroidal Na+/K+ ATPase inhibitor 3-R-POD induces apoptosis, paralleled by altered BCL-2 and c-Fos gene transcription, inhibition of the pro-survival FAK signalling, upregulation of the pro-apoptotic p38 MAPK pathway and stimulation of caspase-3 activity.