Chondromodulin-I (ChM-I) is a 25-kDa glycoprotein in cartilage matrix that inhibits angiogenesis. It contains two distinctive structural domains: the N-terminal third of the molecule is a hydrophilic domain that contains O-linked and N-linked oligosaccharide chains, and the C-terminal two-thirds is a hydrophobic domain that contains all of the cysteine residues. In the present study, we have attempted to further uncover the structural requirements for ChM-I to exert anti-angiogenic activity by monitoring its inhibition of the vascular endothelial growth factor (VEGF)-A-induced migration of HUVEC in vitro. Site-directed mutagenesis experiments revealed that the cyclic structure formed by the disulfide bridge between Cys83 and Cys99 in human ChM-I is indispensable for its anti-angiogenic function. Moreover, the C-terminal hydrophobic tail (from Trp111 to Val120) was found to play an important role in ensuring the effectiveness of ChM-I activity on HUVEC. A synthetic cyclic peptide corresponding to the ChM-I region between Ile82 to Arg100 also inhibited the migration of HUVEC, while replacing the Cys83 and Cys99 residues in this peptide with Ser completely negated this inhibitory activity. An additional synthetic cyclic peptide harboring the hydrophobic C-terminal tail of ChM-I clearly mimicked the inhibitory action of this protein on the migration of HUVEC and successfully inhibited tumor angiogenesis and growth in a xenograft mouse model of human chondrosarcoma. (Cancer Sci 2012; 103: 1311–1318)