Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and suppresses neovascularization and tumor growth. However, the inhibitory mechanism of endostatin in human endothelial cells has not been characterized yet. Electron microscopic analysis revealed that endostatin induced formation of numerous autophagic vacuoles in endothelial in 6 to 24 h after treatment. Moreover, there was only a 2- to 3-fold increase in intracellular reactive oxygen species after endostatin treatment. Endostatin-induced cell death was not prevented by antioxidants (vitamin C, vitamin E, or propyl gallate) or caspase inhibitors, suggesting that the increase of oxidative stress or the activation of caspases may not be the crucial factors in the anti-angiogenic mechanism of endostatin. However, the cytotoxicity of endostatin was significantly reduced by 3-methyladenine (a specific inhibitor of autophagy) and serine and cysteine lysosomal protease inhibitors (leupeptin and aprotinin). Taken together, these results suggest that in human endothelial cells: (1) endostatin predominantly causes autophagic, rather than apoptotic, cell death, (2) endostatin-induced autophagic cell death occurs in the absence of caspase activation and through an oxidative-independent pathway, and (3) endostatin-induced "autophagic cell death" or "type 2 physiological cell death" is regulated by serine and cysteine lysosomal proteases.