Coordinated migration of endothelial cells models the remodeling of existing endothelia as well as angiogenesis and vasculogenesis. Platelet-endothelial cell adhesion molecule-1, PECAM-1, a transmembrane endothelial adhesion protein, binds and activates the tyrosine phosphatase SHP-2 via phosphotyrosines 663 and 686. PECAM-1 phosphorylation and recruitment of SHP-2 are regulated by cell-cell and cell-substrate adhesion. We found that PECAM-1 is dephosphorylated on tyrosine 686 during endothelial migration, resulting in diffuse dispersal of PECAM-1 and SHP-2. Overexpression of native PECAM-1 slowed, and nonphosphorylatable PECAM-1 increased, endothelial migration, implying that the SHP-2-regulatory phosphotyrosines negatively regulate migration. Using differentially phosphorylated recombinant proteins we found that phosphotyrosine 686 preferentially mediates binding and 663 mediates activation of SHP-2 by PECAM-1. In PECAM-1-null endothelial cells, SHP-2 bound and dephosphorylated an alternative set of phosphoproteins and its distribution to the cytoskeletal fraction was significantly decreased. Tyrosine phosphorylation of beta-catenin and focal adhesion kinase was increased in endothelial cells overexpressing nonphosphorylatable PECAM-1. Thus homophilically engaged, tyrosine-phosphorylated PECAM-1 locally activates SHP-2 at cell-cell junctions; with disruption of the endothelial monolayer, selective dephosphorylation of PECAM-1 leads to redistribution of SHP-2 and pro-migratory changes in phosphorylation of cytoskeletal and focal contact components.