Aim To investigate the effect of 5-azacytidine on the differentiation of rat bone marrow mesenchymal stem cells(BMSC) into cardiomyocyte-like cells,and explore the role of fluid shear stress. Methods BMSC were isolated from rats marrow mononuclear cells by attaching growth.The 4 th generation of BMSC were exposed to 3,5,10,15 and 20 μmol/L 5-azacytidine for 12,24 and 48 hours respectively.10 μmol/L for 24 hours as best induction concentration was established based on α-actin expression of cardiomyocyte-like cells after 3 weeks by immunofluorescence.The modles of BMSC were divided into four groups: no fluid shear stress group,load 5 dyn/cm2 fluid shear stress group,load 15 dyn/cm2 fluid shear stress group and load 25 dyn/cm2 fluid shear stress group.After 24 hours,the morphology changes of BMSC was observed by inverted microscopy.RT-PCR was used to examine cTnI mRNA expression after 4 weeks later. Results BMSC gradually became spindle with the passage.5-Azacytidine induced BMSC increases cell bodies and extended long thin processes,branching occurs at the protruding end,some of the processes connecting adjacent cells to form a network,showed morphological transformation to the direction of the characteristics of myocardial cells.α-actin was expressed by inducation after 3 weeks.Through fluid shear stress mechanical stimulation,cTnI expression increased,positive bands was more significant than no fluid shear stress stimulation cells.It was the most obvious in 15 dyn/cm2 fluid shear stress group.However,the change of 25 dyn/cm2 fluid shear stress group was not increased at a positive correlation. Conclusions 5-Azacytidine can induce the differentiation of BMSC into cardiomyocyte-like cells.5-Azacytidine combined with shear stress not only induce the differentiation of BMSC into cardiomyocyte-like cells,but also cause cell differentiation,which seems to have a better effect than only 5-azacytidine.