Limited efficacy of stem cells transplantation for improvement of cardiac function should be overcome. We hypothesized that transplantation of preconditioned stem cells would enhance differentiation and integration of stem cells, resulting in improved cardiac function in myocardial infarction(MI). We tested 3 different conditions for human umbilical cord mesenchymal stem cells(MSC) in rat MI model. At 1 week after MI, rats underwent injection into periinfarct area of culture media(control: n=5), DiI labeled untreated MSC(n=11), pretreated MSC with 5-azacytidine(n=11),and pretreated MSC with myocardial protein obtained from rats with MI and pressure chamber(n=9). The LV systolic function was evaluated with echocardiography at 4 weeks and 8 weeks after MSC injection. Myocardial capillary density, area of infarction, and expression of cardiac markers were evaluated with immunohistochemistry after 8 weeks. LV systolic function of rats injected with pretreated MSC with myocardial protein and oscillating pressure improved at 8weeks more than other groups. Additionally, in treated MSC with myocardial protein, capillary density was significantly higher and expressions of troponin T were markedly increased in transplanted MSC compared to other groups. Preconditioning of MSC with myocardial protein and oscillating pressure enhanced cardiomyogenic differentiation of MSC, angiogenesis, and improvements of cardiac function. Our study suggests that the preconditioning of MSCs with biochemical and physical stimuli would give better results in stem cell therapy.