For successful systemic stem cell therapy, mesenchymal stem cells (MSCs) must transmigrate across the endothelium and invade their target tissue. To date, most of the underlying mechanisms of transmigration and invasion remain to be elucidated. Improving our knowledge on these core processes might elevate the efficiency of stem cell therapy. Our aim was therefore to characterize key mechanisms involved in transmigration and invasion of MSCs. Co-cultivation experiments infer that MSCs integrate into the endothelial monolayer. However, the time course of adhesion, integration and transmigration depends on the endothelial phenotype and is most effective in venous vessels of the myocardium. Thus, a variable capacity for transmigration exists within the vasculature. Additionally, three-dimensional systems reveal that MSCs penetrate the endothelium and invade the surrounding tissue via plasmic podia. Furthermore, transmigration not only requires the interaction of vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4) as verified by blocking experiments, but also triggers the clustering of beta1 integrins. In addition, in situ zymographies infer the activation of gelatinases at sites of MSC invasion into myocardial tissue. As evidenced by ELISA, MSCs secrete matrix metalloproteinase (MMP)-2 but not MMP-9. Finally, media containing additional cytokines accelerate the transmigration. Concluding, key players involved in transmigration and invasion of MSCs are the endothelial phenotype, VCAM-1/VLA-4, beta1 integrins, MMP-2 secretion and cytokines.