Trastuzumab (TZM) significantly improves the outcomes of patients with breast cancer; however, it is associated with severe cardiotoxicity. Ginsenoside Rg2 was reported to exert protective effects against myocardial injury and apoptosis in human cardiomyocytes (HCMs). However, whether ginsenoside Rg2 protects HCMs against TZM-induced toxicity remains unclear. The present study investigated the proliferation of HCMs using a Cell Counting Kit-8 assay and Ki67 immunofluorescence staining. Apoptotic cells were detected by Annexin V/propidium iodide staining and flow cytometry. Furthermore, monodansylcadaverine staining was performed to detect cell autophagy. In addition, western blotting was used to detect the expression levels of phosphorylated (p)-Akt, p-mTOR, beclin 1, microtubule associated protein 1 light chain 3 alpha (LC3) and autophagy protein 5 (ATG5) in HCMs. Pretreatment with ginsenoside Rg2 significantly protected HCMs against TZM-induced cytotoxicity by inhibiting apoptosis. Furthermore, pretreatment with ginsenoside Rg2 induced autophagy in HCMs by upregulating the expression levels of p-Akt, p-mTOR, beclin 1, LC3 and ATG5. The results obtained in the present study suggested that ginsenoside Rg2 could protect HCMs against TZM-induced cardiotoxicity by activating autophagy. Therefore, ginsenoside Rg2 may serve as a potential therapeutic agent to prevent TZM-related cardiotoxicity in patients with breast cancer.