AbstractAn incomplete understanding of the cellular functions and underlying mechanisms of zinc ions released from zinc‐based stents in atherosclerosis (AS) therapy is one of the major obstacles to their clinical translation. The existing evaluation methodology using cell monolayers has limitations on accurate results due to the lack of vascular architectures and pathological features. Herein, the authors propose a 3D biomimetic AS model based on a multi‐layer vascular structure comprising endothelial cells and smooth muscle cells with hyperlipidemic surroundings and inflammatory stimulations as AS‐prone biochemical conditions to explore the biological functions of zinc ions in AS therapy. Concentration‐dependent biphasic effects of zinc ions on cell growth are observed both in cell monolayers and 3D AS models. Nevertheless, the cells within 3D AS model exhibit more accurate biological assessments of the zinc ions, as evidenced by augmented pathological features and significantly higher half‐maximal inhibitory concentration values against zinc ions. Based on such a developed 3D biomimetic AS model, the inhibitory effects on the deoxyribonucleic acid (DNA) synthesis, significantly influenced biological processes like cell motility, proliferation, and adhesion, and several potential bio‐targets of zinc ions of cells are revealed.