Reactive oxygen species (ROS) are associated with inflammation and vasculature dysfunction. This study aimed to investigate the potential role of the ROS on vascular Toll-like receptor 4 (TLR4)-mediated proinflammatory and proliferative phenotype of vascular smooth muscle cells (VSMCs). A wire-induced carotid injury model was used in male TLR4-deficient (TLR4(-/-)) and wild-type C57BL/6J mice to induce neointima formation. In the presence or absence of the ROS scavenger apocynin for 14 days, increased TLR4 and proinflammatory cytokines were observed in wire injury-induced carotid neointima and in platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMCs. The TLR4(-/-) protected the injured carotid from neointimal formation and impaired the cellular proliferation and migration in response to PDGF-BB. Apocynin attenuated intimal hyperplasia. Pre-treatment with apocynin significantly inhibited intracellular ROS generation, accompanied by a significant suppression of TLR4 and proinflammatory cytokines expression, and VSMC proliferation and migration. However, the results were not obvious in TLR4(-/-) condition. These findings highlight the importance of ROS inhibition in TLR4-mediated proinflammatory and proliferative phenotype of VSMCs, and suggest ROS as an essential therapeutic target for TLR4-associated vascular inflammation and vascular diseases.