Podosomes are actin rich adhesion structures capable of extracellular matrix remodeling (ECM) and facilitate invasive cell migration. ECM remodeling and migration of endothelial cells (ECs) are prerequisites for angiogenesis, an important process in development and cardiovascular disease. In this study using a combination of molecular genetics and high resolution microscopy, we have demonstrated that adaptor proteins Nck1 and Nck2, that links signaling by tyrosine phosphorylation with actin dynamics, promote podosome formation and function. Expression of human Nck1-YFP or human Nck2-mCherry induces podosome formation in human umbilical vein endothelial cells. Silencing of Nck by retroviral expression of short hairpin RNA disrupted podosome formation in Src transformed endothelial cells. Both number of cells with podosomes and number of podosomes per cell decreased significantly (P<0.05) compared with control and rescued cells. Functionally, Nck silenced cells were deficient in fluorescent gelatin matrix degradation, an effect that could be almost completely reversed by expression of siRNA-resistant Nck2. Further, overexpression of Nck in Src transformed endothelial cells induced an even higher (p<0.05) index of matrix degradation when compared with Src transformed control cells. Overall, a high degree of colocalization between F-actin and areas of degradation was observed. However, areas of degradation without cellular/F-actin colocalization [[Unable to Display Character: –]] presumably due to the highly motile nature of HUVEC [[Unable to Display Character: –]] were also observed. Mechanistically we found that Nck promote podosome biogenesis through interaction with p62Dok. Collectively, these results provide strong support for a critical role of Nck adaptors in the invasive program of endothelial cells which is important in health and diseases.