The formation of new blood vessels from pre-existing ones requires highly coordinated restructuring of endothelial cells (EC) and the surrounding extracellular matrix. Directed EC migration is a central step in this process and depends on cellular signaling cascades that initiate and control the structural rearrangements. On the basis of earlier findings that ERK5 deficiency in mouse EC results in massive defects in vessel architecture, we focused on the impact of the MEK5/ERK5 signaling pathway on EC migration. Using a retroviral gene transfer approach, we found that constitutive activation of MEK5/ERK5 signaling strongly inhibits EC migration and results in massive morphological changes. The area covered by spread EC was dramatically enlarged, accompanied by an increase in focal contacts and altered organization of actin filaments. Consequently, cells were more rigid and show reduced motility. This phenotype was most likely based on decreased focal contact turnover caused by reduced expression of p130Cas, a key player in directed cell migration. We demonstrate for the first time that ERK5 signaling not only is involved in EC survival and stress response but also controls migration and morphology of EC.