Background: Toll-like-receptors (TLRs) provide a critical link between innate and adaptive immune responses. It has been shown that TLR5 ligand Flagellin can enhance the suppressive capacity of regulatory T-cells (Treg), but can also functions as an adjuvant. The immune response in atherosclerosis is characterized by an imbalance of pro- and anti-atherogenic T-cells. We aimed to establish if the TLR5/Flagellin axis is involved in the immune response of atherosclerosis. Methods: We first assessed the effect of Flagellin exposure on macrophage maturation and T-cell polarization. Next, we created TLR5-/-/LDLr-/- chimeras to study the TLR5/Flagellin axis in atherosclerosis. Results: Flagellin exposure to primary macrophages did not result in clear polarization differences, but we did observe a less migratory phenotype (decreased MCP-1, CCR2 expression) in TLR5-/- macrophages. Interestingly, expression of the T-cell polarizing cytokine IL-6 was induced by Flagellin exposure, a phenomenon not observed in TLR5-/- macrophages. Next, we assessed potential T cell polarizing properties of Flagellin. Flagellin can induce expansion of regulatory T-cells, however this induction is completely overruled when Flagellin is used as an adjuvant. Hematopoietic absence of TLR5 significantly attenuates atherosclerotic lesion formation by 25% (1.03±0.06×106 μm2 vs 0.79±0.06 ×106 μm2 in TLR5-/-, p = 0.01). This was accompanied by a decrease in macrophage area (-46%, p = 0.01) and necrotic core size (-32%, p<0.05) while collagen content was similar between groups. Interestingly, plasma levels of IL-6 were significantly lower in TLR5-/- chimeras (40.2±6.3 in WT vs. 15.1±2.7 pg/ml in TLR5-/-, p=0.003). Concomitantly, TLR5-/- chimeras displayed defective T-cell responsiveness, as seen by impaired proliferation and decreased splenic T cell content. In conclusion, hematopoietic TLR5 deficiency inhibits atherosclerotic lesion formation by attenuated macrophage accumulation and defective T cell responsiveness.