The aim of this study was to investigate the role of endothelial nitric oxide synthase (eNOS) in the host myocardium on bone marrow mesenchymal stromal cells (MSC) migration to the ischemic myocardium and whether stromal cell-derived factor-1alpha (SDF-1alpha) contributes to eNOS-mediated MSC migration. MSCs and coronary microvascular endothelial cells were isolated from adult wild-type (WT) mouse bone marrow and hearts, respectively. Cultured neonatal cardiomyocytes from WT, eNOS(-/-), and eNOS overexpressing transgenic (Tg) mice were subjected to anoxia and reoxygenation (A/R), and the conditioned medium was used as a chemoattractant for in vitro transendothelial migration assay. MSC migration was decreased in the presence of conditioned medium derived from eNOS(-/-) cardiomyocytes but increased in the presence of eNOS-Tg conditioned medium. SDF-1alpha expression was decreased in eNOS(-/-) but increased in eNOS-Tg cardiomyocytes following A/R and in the myocardium following ischemia/reperfusion (I/R). SDF-1alpha expression was cGMP-dependent as inhibition of soluble guanylyl cyclase decreased SDF-1alpha expression in WT cardiomyocytes. MSCs expressed very low levels of eNOS proteins compared with the adult myocardium. To examine MSC migration in vivo, MSCs derived from mice expressing enhanced green fluorescence protein (EGFP(+)) were intravenously administered to WT mice subjected to myocardial I/R. EGFP(+) cells in the ischemic region were decreased in eNOS(-/-) but increased in eNOS-Tg compared with WT hearts. MSC treatment improved cardiac function following I/R in WT but not in eNOS(-/-) mice. In conclusion, eNOS in the host myocardium promotes MSC migration to the ischemic myocardium and improves cardiac function through cGMP-dependent increases in SDF-1alpha expression.