Background: The stimulation of vascular smooth muscle cell (SMC) is critical steps in the development of the neointimal tissues that contribute to the restenosis of an arterial wall. RGD-containing peptides are able to inhibit the binding of ligands to certain beta3 integrins which are involved in neointimal hyperplasia. The purpose of this study was to examine the biological effects of saxatilin, a novel disintegrin from the venom of a Korean snake (Gloydius saxatilis) and RNA interference for specific integrins on vascular smooth muscle cells. Method: To perform this work we first produced large amounts of the active form of recombinant saxatilin in yeast Pichia pastoris and constructed siRNA expression vector for alpha V and alpha 5 integrin subunit. To elucidate the detailed mechanim involved in the inhibitionn of neointimal hyperplasia, the effects of integrin antagonists, an RGD-containing disintegrin saxatilin and RNA interference to integrins, on smooth muscle cell have been studied. Results: Saxatilin interacts with integrin alpha(v)beta(3) and significantly suppresses the adhesion of rat aortic SMC to vitronectin with an IC50 of 65 nM. It disassembled cortical actins at focal adhesions and induced cells to be rounded and detached, but it did not alter microtubule structures in the early stage of cells being rounded. This disassembly of focal adhesion in saxatilin-treated HCASMCs involved caspase-induced paxillin degradation. Focal adhesion kinase(FAK) and ERKs of saxatilin-treated SMCs were temporally phosporylated. Saxatilin affected cell cycle progression of SMCs by increasing CDK inhibitors (p21 and p27) and reducing cyclins (D 1/2 and E). The integrin RNAi to integrin alpha V and alpha 5 treatments were effectively down regulated alpha(v) and alpha(5) on VSMC that disassembled cortical actins at focal adhesions and detached on targeting extracelluar matrix protein. Conclusion: These results may provide new insights into role of integrin in SMC pathophysiology as well as role of SMCs in the process of neointimal hyperplasia and also suggest significant implications for integrin antagonistic therapy of the treatment for restenosis and atherosclerosis.