BACKGROUND: Atherosclerosis (AS) is the leading cause of cardiovascular death worldwide. Macrophage ferroptosis is implicated in AS progression. The precise mechanism by which mixed lineage kinase 3 (MAP3K11, MLK3) regulates macrophage ferroptosis in atherosclerosis is poorly understood. METHODS: From the Gene Expression Omnibus (GEO) database, bulk and single-cell RNA-sequencing (scRNA-seq) datasets from vascular tissues were obtained. MLK3 was discovered to be a crucial ferroptosis-related gene (FRG) implicated in AS by means of differential analysis, weighted gene co-expression network analysis (WGCNA), and a ferroptosis gene set. Macrophages were shown to be the main cell type that expressed MLK3 when immune-infiltration profiling and single-cell RNA sequencing were combined. In vitro, macrophages were treated with Oxidized low-density lipoprotein (ox-LDL) or erastin, with or without MLK3 knockdown. Animal models assessed AS progression, ferroptosis, collagen deposition, and JNK/p53 activation. Immunofluorescence and clinical plasma MLK3 assays were performed. RESULTS: MLK3 was predominantly expressed in plaque macrophages. Ox-LDL upregulated MLK3 and induced ferroptosis, while MLK3 knockdown attenuated ox-LDL-induced ferroptosis by suppressing JNK/p53 signaling, without affecting erastin-induced ferroptosis. Animal models showed increased MLK3 expression, ferroptosis, JNK/p53 activation, plaque area, and collagen deposition during AS progression. Immunofluorescence confirmed MLK3 co-localization with ferroptosis-associated proteins in plaque macrophages. Plasma MLK3 levels were consistently elevated in AS patients. CONCLUSION: MLK3 accelerates the development of atherosclerotic lesions by driving macrophage ferroptosis through JNK/p53 signaling.