Antiangiogenesis represents a promising therapeutic strategy for the treatment of various malignances. Isthmin is a gene highly expressed in the isthmus of the midbrain-hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kDa protein containing a Thrombospondin Type 1 Repeat (TSR) domain in the central region and an Adhesion-associated domain in MUC4 and Other Proteins (AMOP) domain at the C-terminal. In this work, we demonstrate that isthmin is a novel angiogenesis inhibitor. Recombinant mouse isthmin inhibited endothelial cell (EC) capillary network formation on Matrigel through its C-terminal AMOP domain. It also suppressed VEGF-bFGF induced in vivo angiogenesis in mouse. It mitigated VEGF-stimulated EC proliferation without affecting EC migration. Furthermore, isthmin induced EC apoptosis in the presence of VEGF through a caspase-dependent pathway. Isthmin binds to alphavbeta5 integrin on EC surface and support EC adhesion. Overexpression of isthmin significantly suppressed mouse B16 melanoma tumor growth through inhibition of tumor angiogenesis without affecting tumor cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmental vessels in the trunk. Our results demonstrate that isthmin is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis.