Objective To investigate the effect of shear stress combined with lysate of myocardial cells on cardiomyogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods Myocardial cells isolated from neonatal rats were lysed by repeated freezing and defrosting. BMSCs isolated from the marrow of adult rat tibia and femur were randomly assigned to four groups: Group A was BMSCs cultured in complete medium under static state as the control; Group B was BMSCs subjected to laminar shear stress with a parallel plate type device; Group C was BMSCs induced by lysate of myocardial cells; Group D was BMSCs subjected to shear stress combined with lysate of myocardial cells. The cardiomyogenic differentiation of BMSCs was analyzed by RT-PCR, immunocytochemistry and Western blotting. Results Shear stress or/and lysate of myocardial cells treatment induced the expression of cardiomyocyte related markers at both mRNA and protein levels. A combination of shear stress and lysate of myocardial cells had a stronger effect on the expression of cardiomyocyte related markers than either treatment alone. Conclusions Shear stress combined with lysate of myocardial cells treatment could induce BMSC differentiation into cardiomyocytes, which provided an approach to obtain more committed differentiated cells for research or for clinic, and as reference for the design of appropriate biological reactor.