Objective To investigated the effect of Osthole on carotid intimal hyperplasia in rats after balloon injury. Methods Male SD rats were randomly assigned to a sham group, a model group, a low-dose group and a high-dose group. The rats of the model group were injured by balloon catheter. The rats in the low-dose group and the high-dose group were given 10 mg/(kg·d) and 30 mg/(kg·d) respectively for 16 days while those in the sham group and model group were given normal saline. On the 13th day after balloon injury, HE staining was applied for observation of the morphological changes of carotid intima, immunohistochemical staining was used to detect the expression of proliferating cell nuclear antigen (PCNA) in neointima, ELISA was employed to determine the levels of tumor necrosis factor α (TNF-α) and IL-1β, and Western blot was performed to test the expressions of nuclear factor kappa B (NF-κB) and Toll-like receptor (TLR4) proteins. Results Compared with the model group, Osthole significantly decreased the neointima area and the intimal/media ratio, markedly reduced positive index of PCNA expression. Furthermore, Osthole significantly inhibited NF-κB and TLR4 over-expressions induced by balloon injury and reduced the levels of TNF-α and IL-1β. Conclusions These findings suggest that Osthole is involved in the control of neointimal formation via inhibiting TLR4 and NF-κB activation, decreasing inflammatory reaction and inhibiting the proliferation of vascular smooth muscle cells.