Cardiovascular diseases remain the leading causes of death worldwide. Stem cell therapy offers a promising option to regenerate injured myocardium. Among the various types of stem cells, cardiosphere cells represent a mixture of intrinsic heart stem cells and supporting cells. The safety and efficacy of cardiosphere cells have been demonstrated in recent clinical trials. Cell-matrix interaction plays an important role in mediating the engraftment of injected stem cells. Here, we studied the role of integrin β-3 in cardiosphere-mediated cell therapy in a mouse model of myocardial infarction. Our results indicated that inhibiting integrin β-3 reduced attachment, retention and therapeutic benefits of human cardiospheres in mice with acute myocardial infarction. This suggests integrin β-3 plays an important role in cardiosphere-mediated heart regeneration.