Abstract The myocardial microenvironment plays a decisive role in the survival, migration and differentiation of stem cells. We studied myocardial micro-environmental changes induced by ultrasound-targeted microbubble destruction (UTMD) and their influence on the transplantation of mesenchymal stem cells (MSCs). Various intensities of ultrasound were applied to the anterior chest in canines with myocardial infarction after intravenous injection of microbubbles. The expression of cytokines and adhesion molecules in the infarcted area of the myocardium was detected after three sessions of UTMD in 1 wk. Real-time quantitative reverse transcription polymerase chain reaction (RTQ-PCR) showed that the expression of vascular cell adhesion molecule-1 (VCAM-1), stromal cell-derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF) in the 1.5 W/cm2 and 1 W/cm2 groups was markedly increased compared with the 0.5 W/cm2 or the control groups (3.8- to 4.7-fold, p < 0.01), and the expression of interleukin-1β (IL-1β) in the 1.5 W/cm2 group was increased twofold over the 1.0 W/cm2 group, whereas the 0.5 W/cm2 group experienced no significant changes. UTMD at 1.0 W/cm2 was performed as previously described before mesenchymal stem cell (MSC) transplantation. Myocardial perfusion, angiogenesis and heart function were investigated before and 1 month after MSC transplantation. Coronary angiography and 99mTc-tetrofosmin scintigraphy revealed that myocardial perfusion was markedly improved after UTMD + MSCs treatment (p < 0.05). At echocardiographic analysis, heart function and the wall motion score index were significantly improved by UTMD + MSCs treatment compared with MSCs or UTMD alone and the control. In a canine model of myocardial infarction, therapeutic effects were markedly enhanced by MSC transplantation after the myocardial micro-environmental changes induced by UTMD; therefore, this novel method may be useful as an efficient approach for cellular therapy.