Mitoxantrone (MTX) is a chemotherapeutic agent used to treat solid tumors and hematological malignancies, as well as multiple sclerosis. Nevertheless, MTX has been associated with serious adverse side effects, such as cardiotoxicity. Considering that aging is a known risk factor for the development of cardiovascular diseases and cancer, our study aimed to assess the molecular effects of MTX on cardiac muscle of aged mice. Old male CD-1 mice (19 months) received a pharmacologically relevant dose of MTX, consisting of 6 intraperitoneal administrations for three weeks (biweekly) to achieve a cumulative dose of 4.5 mg/kg of MTX (MTX group) or saline (CTRL group). Animal experiments were performed with the approval of the national competent authorities - General Directorate of Food and Veterinary Medicine (DGAV), and mice welfare was monitored daily. Mice were sacrificed two months after the last administration for collection of blood and heart. MTX group showed an increase in serum glucose levels, despite no other differences. Regarding the heart, MTX decreased the content of glucose transporter GLUT4 (GLUT4) and phosphofructokinase (PFKM), suggesting a lower use of glycolysis. Despite no major differences for the mitochondrial biogenesis markers evaluated, the content of autophagy protein 5 (ATG5), atrogin, and CCAAT-enhancer-binding proteins β (CEBPβ) was decreased in the MTX group, highlighting the influence of this drug on cardiac autophagy, proteolysis, and regeneration, respectively. These results suggest that MTX impacts unexplored pathways, so further studies are needed to find new molecular mechanisms that explain the cardiotoxicity induced by this anticancer agent.