Doxorubicin (dox) is an effective chemotherapeutic agent that leads to cardiotoxicity. An alternative treatment for dox-cardiotoxicity is autologous mesenchymal stem cells (MSCs) transplantation. It remains unclear if dox has deleterious effects on MSCs from subjects under chemotherapy, therefore this study aimed to evaluate dox in vivo toxicological effects on ex vivo cultured MSCs, inferring whether autologous transplantation may be an alternative treatment in patients who are exposed to the drug. Wistar rats received either dox or saline. Following treatments, animals were sacrificed and bone marrow MSCs were isolated, characterized for cell surface markers and assessed according to their viability, alkaline phosphatase production, and proliferation kinetics. Moreover, MSCs were primed to cardiac differentiation and troponin T and conexin 43 expressions were evaluated. Compared to control, undifferentiated MSCs from dox group kept the pattern for surface markers and had similar viability results. In contrast, they showed lower alkaline phosphatase production, proliferation rate, and conexin 43 expression. Primed MSCs from dox group showed lower troponin T levels. It was demonstrated a toxic effect of dox in host MSCs. This result renders the possibility of autologous MSCs transplantation to treat dox-cardiotoxicity, which could be a non-suitable option for subjects receiving such antineoplastic agent.