Despite heightened risk of cardiotoxicity associated with combination therapy of anthracyclines and trastuzumab in HER2-positive breast cancer patients, little research effort has been invested in exploring the molecular mechanisms of cardiotoxicity induced by this combination therapy. In this study, we demonstrate that trastuzumab downregulates both gene and protein expressions of type IIB DNA topoisomerase/ DNA topoisomerase IIB (TOP2B), a major intracellular target mediating doxorubicininduced cardiotoxicity, in human primary cardiomyocytes. This in turn induces DNA damage activity and DNA double strand breaks, which is indicated by the enhanced phosphorylation of H2AX (?H2AX) and ataxia telangiectasia and Rad3-related protein (ATR pS428) in trastuzumab-treated cardiomyocytes. Furthermore, concurrent or sequential treatment of doxorubicin and trastuzumab significantly increases the downregulation of the protein levels of TOP2B, enhances apoptosis and cell growth inhibition, and promotes production of reactive oxidative and nitrative species in human cardiomyocytes as compared to either trastuzumab or doxorubicin treatment, indicating augmentation of cardiotoxicity in combination therapy. Additionally, our data reveal that doxorubicin treatment increases the levels of ErbB2/HER2 expression in human cardiomyocytes as compared with that in cells not treated with doxorubicin, leading to the enhanced activity downstream of HER2 signaling. Consequently, this may render the cardiomyocytes to become addicted to HER2 signaling for survival under stressed conditions. Enhanced HER2 protein expression leaves cardiomyocytes more sensitive to trastuzumab treatment after doxorubicin exposure. This study provides molecular basis for significantly increased cardiotoxicity in cancer patients who are treated with anthracyclines and trastuzumab-based combination regimens.