It was reported over a decade ago that tissue inhibitor of metalloproteinases-1 (TIMP-1) suppresses angiogenesis in experimental models but the mechanism is still incompletely understood. This in vitro study focused on the molecular basis of TIMP-1-mediated inhibition of endothelial cell (EC) migration, a key step in the angiogenic process. Both recombinant human TIMP-1 and the synthetic MMP inhibitors, GM6001 and MMP-2-MMP-9 Inhibitor III, suppressed migration of human dermal microvascular endothelial cells (HDMVEC) in a dose-dependent fashion. The MMP-dependent inhibition of migration was associated with increased expression of the junctional adhesion proteins, VE-cadherin and PECAM-1, and VE-cadherin accumulation at cell-cell junctions. TIMP-1 also caused MMP-independent dephosphorylation of focal adhesion kinase (FAK) (pY397) and paxillin, which was associated with reduced number of F-actin stress fibers and focal adhesions. Moreover, TIMP-1 stimulated expression of PTEN that has been shown to reduce phosphorylation of FAK and inhibit cell migration. Our data suggest that TIMP-1 inhibits HDMVEC migration through MMP-dependent stimulation of VE-cadherin and MMP-independent stimulation of PTEN with subsequent dephosphorylation of FAK and cytoskeletal remodeling.