Atherosclerosis (AS) is a chronic inflammatory disease in which macrophages play an indispensable role. Exploration of the effects of aortic cell subpopulations in AS remains challenging due to cellular heterogeneity. Phytosterol oxidation products (POPs) are key dietary factors influencing AS due to their potential pro-inflammatory effects in atherosclerotic mice. However, the contribution of alterations in cellular heterogeneity to this outcome and the exact mechanisms remain elusive. Here, we constructed a novel single-cell transcriptomic landscape of arteries in ApoE^−/− mice fed an atherosclerotic diet without or with POPs. Combining single-cell RNA sequencing (scRNA-seq) with in vitro functional validation, we demonstrated that 7-ketositosterol, a major component of POPs, induced macrophages to skew the pro-inflammatory (M1) phenotype through the TLR4-IRF5 axis, thereby amplifying the inflammatory response. Notably, we verified the presence of this pro-inflammatory immune niche with the same molecular features using publicly available human arterial scRNA-seq data. This demonstrates that this is a reproducible characteristic in human AS. Our study shifts the current paradigm of exploring the biological effects of food components, and provides unprecedented perspectives for the application of single-cell technology to food nutrition research.