OBJECTIVE The aim of this work was to investigate the immunological effect of MENK by analyzing the protein spectrum and bioinformatics of macrophage RAW264.7, and to explore the relationship between macrophage and ferroptosis. RESULTS We employed proteomic analysis to identify differentially expressed proteins (DEPs) between macrophages and macrophages intervened by MENK. A total of 208 DEPs were identified. Among these, 96 proteins had upregulated expression and 112 proteins had downregulated expression. Proteomic analysis revealed a significant enrichment of DEPs associated with iron metabolism. The identification of hub genes was conducted using KEGG pathway diagrams and protein-protein interaction (PPI) analysis. The hub genes identified in this study include HMOX1 and Ferritin (FTH and FTL). A correlation was established between HMOX1, FTH, and FTL in the GO and KEGG databases. The results of PCR, WB and immunofluorescence showed that MENK downregulated the level of HMOX1 and FTH. CONCLUSIONS MENK had the potential to become an adjuvant chemotherapy drug by regulating iron metabolism in macrophages, reducing levels of HMOX1 and ferritin. We proposed an innovative research direction on the therapeutic potential of MENK, focusing on the relationship between ferroptosis and macrophage activity.