Objectives To investigate the protective effect of thrombopoietin (TPO) on myocardial cells in vitro. Methods H9C2 cell line was maintained in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% calf serum. Beating cells from heart ventricles of neonatal heart were cultured at an in vitro system. Apoptosis of the cell line above was induced by treatment of doxorubicin (DOX) and was blocked by TPO. Cell survival rate of H9C2 cell was measured by the MTT assay. Changes of beating rate of neonatal myocardial cells were captured by digital camera and beating rate was calculated. Flow cytometry was employed to study anti-apoptotic effect of TPO by staining JC-1 protein to H9C2 cell. Results MTT assay demonstrated that doxorubicin reduced cell survival rate by 73.8%±1.1%, 50 ng·mL-1 and 100 ng·mL-1 TPO increased cell survival rate by 84.6%±3.6% (P<0.05), 86%±4% (P<0.01) at a dose-dependent manner. Beating rate of primary neonatal myocardial cells also decreased to 15%±8% at 48 h, 100 ng·mL-1 TPO improved beating rate to 48%±11% (P<0.01). TPO decreased apoptotic rate from 19%±9% to 11%±6% (P<0.05). Conclusions TPO has protective effect on myocardial cells in vitro. Anti-apoptosis is one of the mechanisms by which TPO protects injured heart. Objectives To investigate the protective effect of thrombopoietin (TPO) on myocardial cells in vitro. Methods H9C2 cell line was maintained in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% calf serum. Beating cells from heart ventricles of neonatal heart were cultured at an in vitro system. Apoptosis of the cell line above was induced by treatment of doxorubicin (DOX) and was blocked by TPO. Cell survival rate of H9C2 cell was measured by the MTT assay. Changes of beating rate of neonatal myocardial cells were captured by digital Flow cytometry was used to study anti-apoptotic effect of TPO by staining JC-1 protein to H9C2 cell. Results MTT assay demonstrated that doxorubicin reduced cell survival rate by 73.8% ± 1.1%, 50 ng · mL -1 and 100 ng · mL-1 TPO increased cell survival rate by 84.6% ± 3.6% (P <0.05), 86% ± 4% (P <0.01) also decreased to 15% ± 8% a TPO improved beating rate from 48% ± 11% (P <0.01) at 48 h, 100 ng · mL-1. Conclusions TPO decreased from 19% ± 9% to 11% ± 6% has protective effect on myocardial cells in vitro. Anti-apoptosis is one of the mechanisms by which TPO protects injured heart.