The mesenchymal stem cells from Wistar rats adipose tissue was separated and cultured. The passage 3 cells were treated with 10 μmmol/L 5-Azacytidine for 24 h, and cultured in vitro for 1, 2, 3 weeks, then cardiomyocyte protein and cardiogenesis-related gene were deteted by immumohistochemistrical staining. After 1 week, the cells were found to express BMP2, after 2 week, the cells were found to express α-sacromeric actin, Cx43, Nkx2.5, GATA-4 were expressed in the cells and the BMP2 was expressed more, the 5 proteins (α-sacromeric actin, Cx43, BMP2, Nkx2.5, GATA-4 were expressed more in the cells after 3 week,but the α-skeletal myosin was not expressed at all times. The result showed that during differentiating into cardiomyocyte in vitro, there was close relation among the adipose tissue mesenchymal stem cells, cardiogenesis-related genes expression, culturing time and differentiation effect, and there was a few same path between cardiomyocyte differentiation in vitro and cardiac muscle development.