Doxorubicin (DOX) is an effective chemotherapeutic drug used for the treatment of a variety of malignancies. Unfortunately, time and dose-dependent DOX therapy induces cardiotoxicity and heart failure. We previously reported that transplanted embryonic stem (ES) cells and the conditioned medium (CM) can repair and regenerate injured myocardium in acute DOX-induced cardiomyopathy (DIC). However, the effectiveness of ES cell and CM therapeutics has not been challenged in the chronic DIC model. To this end, the long-term impact of ES cells and CM on apoptosis, fibrosis, cytoplasmic vacuolization, oxidative stress, and their associated mediators were examined. Four weeks post-DIC, ES cells and CM-transplanted hearts showed a significant decrease in cardiac apoptotic nuclei, which was consequent to modulation of signaling molecules in the Akt pathway including PTEN, Akt, and mTOR. Cytoplasmic vacuolization was reduced following treatment with ES cells and CM, as was cardiac fibrosis, which was attributable to downregulation of MMP-9 activity. Oxidative stress, as evidenced by DHE staining and lipid peroxide concentration, was significantly diminished, and preservation of the antioxidant defense system was observed following CM and ES cell transplantation. In conclusion, our data suggest that transplanted ES cells and CM have long-term potentiation to significantly mitigate various adverse pathological mechanisms present in the injured chronic DIC heart.