We investigated, whether MSC may influence endothelial function via paracrine mechanisms to facilitate their transmigration through the endothelial barrier. We performed time-dependent (5–20 min) measurements of the intracellular NO-levels (diaminofluorescein, DAF) and immunohistochemical measurements of eNOS-translocation and -phosphorylation as well as phosphorylation of AKT/protein kinase B and ERK in human umbilical endothelial cells (HUVEC) in the presence of endothelial cell growth medium (ECG-medium), or a-MEM medium (aMEM_CON) as well as MSC-preconditioned a-MEM medium (a+MSC).Only after application of a+MSC, a sig. increase in DAF-fluorescence paralleled by a transient increase of eNOS-translocation and eNOS serine 1177-phosphorylation paralleled by an AKT-phosphorylation was observed. In addition, an increase in eNOS-threonine 495 phosphorylation was measured in parallel with an increase in ERK-phosphorylation. ENOS-Ser116 phosphorylation was not altered. Conclusions: MSC emit paracrine factors that alter eNOS-activity. These alterations may be of importance regarding the opening of the endothelial barrier as prejudice for the MSC-transmigration after intravasal application.