Therapeutic manipulation of angiogenesis, the formation of new vascular sprouts from existing capillaries, is one of the promising strategies for treatment of human diseases such as cancer, arthritis, and cardiovascular disorder. In the present study, we examined the effects and molecular mechanism of tissue inhibitor of metalloproteinases-2 (TIMP-2) and endostatin on fibroblast growth factor-2 (FGF-2)-stimulated endothelial cell proliferation, migration and adhesion in vitro, and angiogenesis in vivo. TIMP-2 and endostatin showed potent anti-angiogenic activity in vitro and in vivo. These effects appear to be mediated through different angiogenic signaling pathways. Collectively, our findings demonstrate that TIMP-2 and endostatin strongly inhibit FGF-2-induced angiogenic responses, and the establishment of fast and reproducible evaluation system in vitro and in vivo for the development of anti-angiogenic biomaterials and therapeutics.