Background and Objectives: DW6386 is a new class of potential antitumor compound. DW6386 showed effect on the cytoskeleton similar to those of the antineoplastic drug like paclitaxel. In vitro, we investigated its effects on rat aortic vascular smooth muscle cell (VSMC) proliferation. In vivo, we tested whether perivascular delivery of DW6386 can prevent neointimal formation in a rat carotid artery injury model. Methods and Results: DW6386 potently inhibited the growth of rat aortic VSMCs induced by 5% fetal bovine serum (FBS) and 50 ng/ml platelet-derived growth factor (PDGF)-BB in a dose-dependent manner. DW6386 also strongly inhibited the DNA synthesis induced by FBS and PDGF-BB in a dose-dependent manner. In order to elucidate the inhibitory mechanism of VSMCs growth, cell cycle progression, apoptosis and signaling pathway were also investigated. DW6386 shows no effect on FBS- and PDGF-BB-induced intracellular early signal transductions such as extracellular response kinases 1 and 2 (ERK1/2), phosphatidylinositol 3'-kinase (PI3'-K)/Akt and phospholipase C-款1 (PCL-款1). DW6386 blocked the FBS- and PDGF-BB-induced progression through G₀/G₁ to S phase of the cell cycle in synchronized cells without apoptosis. The expression of p27^Kip1 in PDGF-BB-stimulated VSMCs inactivated cdk2 leading to G₁ growth arrest. Fifteen days after balloon injury, carotid arteries were perfusion-fixed and morphometric analysis was performed. DW6386-treated group showed a significant neointimal formation reduction versus the control group (0.17 짹 0.02 mm² versus 0.22 짹 0.04 mm², p<0.05). Conclusion: This study suggests that DW6386 may inhibit the proliferation of rat aortic VSMC proliferation by perturbing cell cycle progression, which may be due to the activation of p27^Kip1 pathway. We also have demonstrated that perivascular application of DW6386 provides an effective means of inhibiting proliferative response to vascular injury in the rat.