Rapamycin and its analogs (rapalogs), those are the mTOR inhibitor, exhibit immunosuppressant and antitumor activity, and extend survival of patients with metastatic renal-cell carcinoma (mRCC), advanced breast cancer and Pancreatic Neuroendocrine Tumour (PNET). Drug Instructions of Everolimus and Temsirolimus mention that patients taking any medicines for heart conditions or high blood pressure might need to be prescribed a different medicine or the dose of rapalogs may need to be changed. However, whether rapamycin induced cardiotoxicity and the mechanisms need further disclosed. Here, we showed that the primary cultured neonatal rat cardiac myocytes viability was reduced significantly after rapamycin treatment for 72 h. On the other hand, the cells with 3- Methyladenine (3-MA, an autophagy inhibitor) pre-treatment increased cell survival rate compared with rapamycin treatment alone. These results suggest that autophagy plays an important role in rapamycin-mediated primary cultured neonatal rat cardiac myocytes death.