Heart disease is a leading cause of death in newborns and in adults. Efforts to promote cardiac repair by introduction or recruitment of exogenous stem cells hold promise but typically involve isolation and introduction of autologous or donor progenitor cells. We have found that the G-actin-sequestering peptide thymosin beta4 promotes myocardial and endothelial cell migration in the embryonic heart and retains this property in postnatal cardiomyocytes. Survival of embryonic and postnatal cardiomyocytes in culture was also enhanced by thymosin beta4. We found that thymosin beta4 formed a functional complex with PINCH and integrin-linked kinase (ILK), resulting in activation of the survival kinase Akt/PKB, which was necessary for thymosin beta4's effects on cardiomyocytes. After coronary artery ligation in mice, thymosin beta4 treatment resulted in upregulation of ILK and Akt activity in the heart, enhanced early myocyte survival, and improved cardiac function. These findings suggest that thymosin beta4 promotes cardiomyocyte and endothelial migration, survival, and repair and may be a novel therapeutic target in the setting of acute myocardial damage.