Proteolytic enzyme-mediated degradation of the extracellular matrix (ECM) is crucial for the formation of both tumor metastasis and angiogenesis. Recently, several reports have suggested that aminopeptidases are involved in this process, but precisely how is largely unknown. We found here that aminopeptidase N (APN/CD13) was selectively expressed in vascular endothelial cells including human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC), and was not detectable in a majority of normal cells and tumor cell lines we examined. RNA interference (RNAi) of APN resulted in the inhibition of capillary tube formation of HUVEC on Matrigel. APN siRNA suppressed the migration of HUVEC through a fibronectin-coated Transwell membrane, and reduced the cellular adhesion to Matrigel and various adhesion molecules including type IV collagen, type I collagen and fibronectin. These findings suggest that APN is a multifunctional protein with important roles in vascular endothelial morphogenesis during angiogenesis.