Thrombospondin-1 (TSP1) is a potent natural inhibitor of angiogenesis. Although TSP1 has been reported to induce endothelial cell apoptosis in vitro and to downregulate neovascularization in vivo, the molecular mechanisms that link these two processes have yet to be established. Here we report that TSP1 mediates endothelial cell apoptosis and inhibits angiogenesis in association with increased expression of Bax, decreased expression of Bcl-2, and processing of caspase-3 into smaller proapoptotic forms. The ability of TSP1 to induce both endothelial cell apoptosis in vitro and to suppress angiogenesis in vivo was blocked by the caspase-3 inhibitor z-DEVD-FMK. TSP1 also attenuated VEGF-mediated Bcl-2 expression in endothelial cells in vitro and angiogenesis in vivo. Furthermore, TSP1 induced endothelial cell apoptosis and inhibited neovascularization in sponge implants in SCID mice. We conclude that TSP1 induces endothelial cell apoptosis and inhibits neovascularization by altering the profile of survival gene expression and activating caspase-3.