The beneficial effects of stem cells in clinical applications to date have been modest, and studies have reported that poor engraftment might be an important reason. As a strategy to overcome such a hurdle, we developed the spheroid three dimensional (3D) bullet as a delivery method for human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) through the maintenance of cell-cell interactions without additional xenofactors, cytokines, or matrix. We made spheroid 3D-bullets from hUCB-MSCs at 24 hours' anchorage-deprived suspension culture. To investigate the in vivo therapeutic efficacy of 3D-bullets, we used rat myocardial infarction (MI) model. Transplantation of 3D-bullet was better than that of single cells from monolayer culture or from 3D-bullet in improving left ventricular (LV) contractility [LV ejection fraction (LVEF) or LV fractional shortening (LVFS)] and preventing pathologic LV dilatation [LV end-systolic diameter (LVESD) or LV end-diastolic diameter (LVEDD)] at 8 weeks. In the mechanism study of 3D-bullet formation, we found that calcium-dependent cell-cell interaction was essential and that E-cadherin is a key inducer mediating hUCB-MSC 3D-bullet formation among several calcium-dependent adhesion molecules which were nominated as candidates after cDNA array analysis. In more specific experiments with E-cadherin overexpression using adenoviral vector or with E-cadherin neutralization using blocking antibody, we found that E-cadherin regulates vascular endothelial growth factor (VEGF) secretion via extracellular signal-regulated kinase (ERK)/v-akt murine thymoma viral oncogene homolog1 (AKT) pathways. During formation of spheroid 3D-bullets, activation of E-cadherin in association with cell-cell interaction turns on ERK/AKT signaling pathway that are essential to proliferative and paracrine activity of MSCs leading to the enhanced therapeutic efficacy.