Severe tissue necrosis with a retarded wound healing process is a major symptom of a cobra snakebite. Cardiotoxins (CTXs) are major components of cobra venoms that belong to the Ly-6 protein family and are implicated in tissue damage. The interaction of the major CTX from Taiwan cobra, i.e. CTX A3, with sulfatides in the cell membrane has recently been shown to induce pore formation and cell internalization and to be responsible for cytotoxicity in cardiomyocytes (Wang, C.-H., Liu, J.-H., Lee, S.-C., Hsiao, C.-D., and Wu, W.-g. (2006) J. Biol. Chem. 281, 656-667). We show here that one of the non-cytotoxic CTXs, i.e. CTX A5 or cardiotoxin-like basic polypeptide, from Taiwan cobra specifically bound to alpha(v)beta3 integrin and inhibited bone resorption activity. We found that both membrane-bound and recombinant soluble alpha(v)beta3 integrins bound specifically to CTX A5 in a dose-dependent manner. Surface plasmon resonance analysis showed that human soluble alpha(v)beta3 bound to CTX A5 with an apparent affinity of approximately 0.3 microM. Calf pulmonary artery endothelial cells, which constitutively express alpha(v)beta3, showed a CTX A5 binding profile similar to that of membrane-bound and soluble alpha(v)beta3 integrins, suggesting that endothelial cells are a potential target for CTX action. We tested whether CTX A5 inhibits osteoclast differentiation and bone resorption, a process known to be involved in alpha(v)beta3 binding and inhibited by RGD-containing peptides. We demonstrate that CTX A5 inhibited both activities at a micromolar range by binding to murine alpha(v)beta3 integrin in osteoclasts and that CTX A5 co-localized with beta3 integrin. Finally, after comparing the integrin binding affinity among CTX homologs, we propose that the amino acid residues near the two loops of CTX A5 are involved in integrin binding. These results identify CTX A5 as a non-RGD integrin-binding protein with therapeutic potential as an integrin antagonist.