Abstract: AIM: To investigate the transdifferentiation of bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes and the temporal expression of regulatory genes in the simulated myocardial microenvironment in which MSCs were co-cultured with cardiomyocytes by indirect contact in vitro and to screen some important genes in this differentiation. METHODS: MSCs isolated from adult rats were cocultured with CMs obtained from neonatal rat ventricles at a 1∶5 ratio in a dual-chamber dish separated by a semipermeable membrane for 2 weeks. During this indirect contact coculture procedure, cardiomyogenic differentiation was confirmed by phase contrast microscope and immunostaining against α-actin and cardiac troponin-T (cTnT), and reverse transcriptase-polymerase chain reaction was performed for temporal expression of regulatory genes TGF-β, Nkx-2.5, GATA-4, MEF-2C and TEF-1. RESULTS: After coculture with CMs, MSCs showed a stick-like or elliptical morphology. Immunofluorescent stain results revealed that α-actin- and cardiac troponin T (cTnT)-positive cells were found in MSCs at day 5 after coculture with CMs, and 29.63% of MSCs were positive for α-actin and 27.38% for cTnT at 14 days. RT-PCR demonstrated that the expression level of TGF-β, Nkx-2.5, GATA-4 and MEF-2C genes began to increase at day 1 after coculture, reached their peak at day 7 and remained relatively high afterwards, even though there was a slight decrease. Only insignificant changes were observed in the expression level of TEF-1 gene in the coculture. CONCLUSION: Indirect contact with CMs (1∶5) is conducive for MSCs to differentiate into CMs in vitro. TGF-β, Nkx-2.5, GATA-4 and MEF-2C genes may play a crucial role during transdifferentiation.