Abstract: The aim of this study is to investigate the impact on the TLR4/MyD88/NF-κB signaling pathway in gypenosides（GPs） anti-atherosclerosis（AS） process of rats, and to predict the active ingredient in GPs anti-atherosclerosis effect tentatively. Sixty SD rats were divided into control group（Control, n=10）, model group（Model, n=10）, simvastatin group（5 mg/kg, Intragastric administration, 10 weeks, n=10）, low-dose GPs group, medium-dose GPs group and high-dose GPs group（40,80,160 mg/kg, intragastric administration, 10 weeks, n=10）, randomly. The control group were fed with a basal diet, and the other groups were treated with a high fat diet（HFD） and vitamin D₃ injection to establish AS model. After 10 weeks, the aortic was detected by hematoxylin-eosin（HE） staining, the tumor necrosis factor-α（TNF-α） level was tested with enzyme-linked immunosorbent assay. Protein of TLR4, MyD88 and NF-κB were measured by Western blot, and mRNA expression level of TLR4, MyD88, and NF-κB were measured by real-time fluorescence quantitative PCR（RT-qPCR） to evaluate the affinity between 3 GPs and TLR4/MyD88/NF-κB signaling pathway respectively by molecular docking. The results showed that pathological changes in the aorta were alleviated after the GPs treatment. The protein and mRNA expression of TLR4, MyD88, NF-κB in the aorta of the rats in the treatment groups showed that the down-regulated trend compared with the model group, the groups treated with high dose GPs（160 mg/kg） significantly down-regulated（P＜0.05）, and the level of proinflammatory mediator TNF-α in serum was also down-regulated compared with the model group, the groups treated with high dose GPs significantly showed the reduced levels of TNF-α（P＜0.05）. The molecular docking showed that GPs XLIX keeped the highest affinity to TLR4/MyD88/NF-κB out of 3 gypenoside ingredients, the TLR4 and NF-κB-related receptor might be its targets. To sum up, TLR4/MyD88/NF-κB inflammatory pathway was interfered in the GPs anti-AS process, and GPs may be against AS by reducing the vascular inflammatory response via inhibiting the expression of TLR4 and its downstream signaling molecules and gypenoside XLIX might be an important component.