[Aims] Inflammatory and proliferative process is one of the major pathophysiologic mechanisms of neointimal hyperplasia following vascular injury. Although sulfasalazine has been used as an anti-inflammatory and immune-modulatory agent in various inflammatory diseases, the therapeutic effect on vascular disease and its main targets have yet to be determined. We investigated whether sulfasalazine could suppress the vascular smooth muscle cell (VSMC) growth, leading to prevention of neointimal hyperplasia. [Methods and results] In cultured VSMCs, we found that sulfasalazine possessed pro-apoptotic and anti-proliferative actions. Unexpectedly, these effects were not mediated by NF-觀B inhibition which has been suggested as anti-inflammatory mechanism of sulfasalazine, but by cell cycle arrest of VSMCs which was mediated by induction of heme oxygenase-1 (HO-1) followed by increased expression of p21waf1/Cip1. The underlying mechanism for sulfasalazine-induced HO-1 expression was through reactive oxygen species (ROS)-dependent nuclear translocation and activation of Nrf2. In a rat carotid artery balloon injury model, administration of sulfasalazine significantly suppressed neointimal growth. In a series of reverse experiments, inhibition of HO-1 by shRNA, ROS by NAC, or Nrf2 by dominant-negative Nrf2 abrogated the beneficial effects of sulfasalazine. [Conclusion] Our data demonstrate that sulfasalazine inhibits in vitro and in vivo VSMC proliferation through a novel signaling pathway and may be a promising therapeutic option to treat proliferative vascular disease.