Background <p>Macrophage migration inhibitory factor (MIF) promotes inflammation, regulates immune responses and chemotherapy resistance in the tumor microenvironment. However, its mechanism of action in colorectal cancer (CRC) metabolic reprogramming and targeted therapeutic potential remain unclear. This study aims to investigate the function, mechanism, and targeted therapeutic potential of MIF in CRC.</p> Methods <p>Data were integrated from TCGA, GTEx, CPTAC, and HPA databases with clinical sample validation. Single-cell sequencing analysis (datasets GSE166555 and GSE144735) was performed, alongside functional assays and mechanistic studies. A novel high-potency MIF inhibitor was identified through virtual screening and validated in vitro and in vivo.</p> Results <p>MIF expression was found to be significantly elevated in CRC tissues and cell lines, correlating with poor overall survival (OS) and disease-specific survival (DSS). Single-cell sequencing confirmed malignant epithelial cells as the primary MIF source. Functional assays demonstrated that MIF knockout suppressed CRC cell proliferation, migration, and tumor growth in vivo, while MIF overexpression promoted these effects. Mechanistically, MIF binds CD74 to upregulate glycolytic enzymes (HK2, PKM2, LDHA), enhancing glucose uptake and lactate/pyruvate production, thereby driving the Warburg effect and CRC progression. Virtual screening identified a novel high-potency MIF inhibitor, F3277-0933 (IC50 = 8.284 μM). In vitro and in vivo, F3277-0933 surpassed the classical inhibitor ISO-1 in suppressing MIF-driven glycolytic reprogramming and proliferation.</p> Conclusion <p>This study elucidates a novel mechanism by which the MIF-CD74 axis drives CRC progression through glycolytic reprogramming and provides robust preclinical evidence for developing MIF-targeted therapies.</p> Graphical Abstract <p></p>