Abstract: Objective To study which kind of cells in myocardium playing determining role in cardiomyogenic differentiation of marrow derived-mesenchymal stem cells (MSCs). Methods Rat marrow derived-MSCs, cardiomyocytes (CM) and endothelial cells (EC) were isolated and cultured, then MSCs were incubated with the CM or EC or cultured alone after labeled by 5-Bromo-2-deoxyuridine (BrdU) for 72 hours. The cells were identified by morphology, immunocytochemistry and double immunocytochemistry. Results More than 95% of the cells isolated and cultured belonged to MSCs, CM or EC. Incubated with CM, most MSCs labeled by BrdU showed cardiomyocyte-like shape. After 4 weeks of cultivation, positive staining of double immunocytochemistry of BrdU and Sarcomeric Actin or Connexin-43 (CX-43) were observed on 44% cells. However, there was no obvious change in morphology and no positive staining of double immunocytochemistry on MSCs incubated with EC or cultured alone after 1、2、3 and 4 weeks of cultivation. Conclusion MSCs can differentiate into cardiomyogenic cells under cardiomyocyte microenvironment in vitro, and cardiomyocytes may play a crucial role for the differentiation.