Endostatin is a potent endogenous angiogenesis inhibitor that induces regression of tumors in mice. Neither an extracellular receptor for endostatin nor intracellular signals that result in the regression of tumor vascular beds have been identified. We demonstrate that endostatin, but not angiostatin, at comparable concentrations to those used in in vivo animal trials, rapidly down‐regulates many genes in exponentially growing endothelial cells. These include immediate early response genes, cell cycle‐related genes, and genes regulating apoptosis inhibitors, mitogen‐activated protein kinases, focal adhesion kinase, G‐protein‐coupled receptors mediating endothelial growth, a mitogenic factor, adhesion molecules, and cell structure components. Suppression of both apoptosis inhibitors and cell proliferation genes may have a limited contribution to the antiangiogenesis process because endostatin induces neither apoptosis nor growth inhibition, unless studied under reduced serum conditions. In contrast, the antimigratory effect of endostatin was rapid and potent even under serum‐supplemented conditions. Endostatin caused gene suppression and migration arrest exclusively in endothelial cells, most profoundly in microvascular endothelial cells. The c‐myc null fibroblasts obtained by targeted homologous recombination showed an attenuated migration rate compared with isogenic parental cells, whereas the introduction of the c‐myc gene into endothelial cells abrogated the antimigratory effect of endostatin. Inhibition of E‐box‐driven transcription by overexpressing max or mad suppressed endothelial migration. Thus, rapid down‐regulation of genes by endostatin neither restores proliferating endothelial cells to their resting states nor induces apoptosis; rather, it potently inhibits endothelial cell migration partly via suppression of c‐myc expression.—Shichiri, M., and Hirata, Y. Antiangiogenesis signals by endostatin. FASEB J. 15, 1044–1053 (2001)