Rationale Accumulating data supports a therapeutic role for mesenchymal stem cell (MSC) therapy; however, there is no consensus on the optimal route of delivery. Objective We tested the hypothesis that the route of MSC delivery influences the reduction in infarct size (IS) and improvement in left ventricular ejection fraction (LVEF). Methods and Results We performed a meta-analysis investigating the effect of MSC therapy in acute myocardial infarction (AMI) and chronic ischemic cardiomyopathy (ICM) preclinical studies (58 studies; n=1165 mouse, rat, swine) which revealed a reduction in IS and improvement of LVEF in all animal models. Route of delivery was analyzed in AMI swine studies and clinical trials (6 clinical trials; n=334 patients). In AMI swine studies, transendocardial stem cell injection (TESI) reduced IS (n=49, 9.4% reduction 95%CI −15.9, −3.0), whereas intramyocardial injection (DI), intravenous infusion (IV), and intracoronary infusion (IC) indicated no improvement. Similarly, TESI improved LVEF (n=65, 9.1% increase 95%CI 3.7, 14.5), as did DI and IV, while IC demonstrated no improvement. In humans, changes of LVEF paralleled these results, with TESI improving LVEF (n=46, 7.0% increase 95%CI 2.7, 11.3), as did IV, but again IC demonstrating no improvement. Conclusions MSC therapy improves cardiac function in animal models of both AMI and ICM. The route of delivery appears to play a role in modulating the efficacy of MSC therapy in AMI swine studies and clinical trials, suggesting the superiority of TESI due to its reduction in IS and improvement of LVEF, which has important implications for the design of future studies.