Human epidermal growth factor receptor-2 (HER2) is overexpressed in up to 30% of breast cancer cases, causing a more aggressive tumour growth and poor prognosis. Trastuzumab, the humanized antibody targeted to HER2, increased the life expectancy of patients, but severe cardiotoxicity emerged as a long-term adverse effect. Clinical evidence highlights that Trastuzumab-induced cardiotoxicity drastically increases in association with Doxorubicin; however, the exact mechanisms involved remain incompletely understood. In order to analyse the molecular mechanisms involved and the possible adaptative responses to Trastuzumab and Doxorubicin treatment, in this study, H9c2 cardiomyoblasts were used. Results showed that Trastuzumab and Doxorubicin sequential administration in cardiomyoblast increased cytosolic and mitochondrial ROS production, intracellular calcium dysregulation, mitochondrial membrane depolarization, and the consequent apoptosis, induced by both Trastuzumab and Doxorubicin alone. Furthermore, in these conditions, we observed increased levels of Connexin43 phosphorylated on Ser368 (pCx43). Since phosphorylation on Ser368 decreases gap junction intracellular communication, thus reducing the spread of death signals to adjacent cells, we hypothesized that the increase in pCx43 could be an adaptative response implemented by cells to defend neighbouring cells by Trastuzumab and Doxorubicin sequential administration. However, the other side of the coin is the resulting conduction abnormalities.