Background & Aim: The forkhead transcription factors are known to suppress growth and promote apoptosis of vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). Cysteine-rich angiogenic protein 61 (CYR61) was cloned as an immediate early gene expressed in fibroblasts on growth factor stimulation and its expression has been associated with vascular restenosis. We tested whether FOXO3a could inhibit neointimal hyperplasia by suppression of CYR61 in balloon injury model. Methods: To evaluate forkhead transcription factor effect on CYR61 expression, VSMCs and HUVECs were infected with adenoviral vectors expressing constitutively active FOXO3a (FOXO3a-TM) and gene expressions were evaluated. Relation between FOXO3a and CYR61 was confirmed by CYR61 reporter plasmid. In vivo experiment, the carotid artery of rat was denuded and adenoviral vectors (Adv-FOXO3a-TM vs GFP) were delivered and degree of neointimal hyperplasia was evaluated. And immunohistochemistry for CYR61 was performed. Results: 1) constitutive activation of FOXO3a signaling suppressed CYR61 mRNA expression both in HUVECs and VSMCs and this suppression was confirmed by Western blot. Cotransfection of Adeno-FOXO3a-TM with the reporter plasmid resulted in a significant decreased in luciferase expression. 2) After injury, immunoblot for phospho-Fox increased from 3 hours and sustained until 24 hours. This suggests during early stage of injury Fox remained in cytoplasm resulting cell proliferation. In FOXO3a-TM delivered group, even in early stage of injury, FOXO3a was located in nucleus and after 14 days, there was a significantly reduced neointimal area (0.16짹0.03 versus 0.05짹0.03mm2, P<0.01) compared with GFP-transfected group. Regarding CYR61 expressions after injury, CYR61 expressions increased. However, in FOXO3a-TM group, CYR61 expressions were much less compared with control. Conclusion: We have shown that FOXO3a suppressed CYR61 expressions both in HUVECs and VSMCs. And Adenoviral gene transfer of the constitutively-active FOXO3a gene significantly inhibited neointimal hyperplasia by suppressing CYR61 expressions. These data would provide important therapeutic implications to control restenosis after vascular interventions.