The present study was performed to examine the protective effects of rhamnetin against the development of atherosclerosis and its effects on the regulation of several pathways. The anti-atherosclerosis effect of rhamnetin was examined in cells stimulated with oxidized low-density lipoprotein (ox-LDL) by estimating the percentages of foam cell formation and apoptotic cells and determining the expression of specific proteins. As an in vivo model of atherosclerosis, apolipoprotein E-deficient (ApoE-/-) mice were treated with intragastric rhamnetin at 100 or 200 mg/kg for 8 weeks. The levels of inflammatory cytokines and oxidative stress parameters were determined in the aortic tissue of rhamnetin-treated ApoE-/- mice. The effects of rhamnetin on the Toll-like receptor 4 (TLR-4) pathway were assessed by quantitative reverse transcription polymerase chain reaction. The results of this study suggest that rhamnetin reduces the percentages of foam cells and apoptotic cells and the expression of CD36 and TLR-4 protein in ox-LDL-stimulated macrophages. The rhamnetin treatment group showed reductions in the lipid profile and levels of parameters of liver function compared to ApoE-/- mice. The levels of inflammatory cytokines and oxidative stress were reduced in the aortic tissue of the rhamnetin treatment group compared to ApoE-/- mice. Treatment with rhamnetin ameliorated the expression of TLR-4 mRNA and components of the TLR-4 pathway in the aortic tissue of ApoE-/- mice. In conclusion, the results of this study suggest that rhamnetin treatment inhibits the inflammatory and pro-atherosclerosis pathways in ApoE-/- mice.