Activation of epidermal growth factor receptor (EGFR) is associated with many human tumours, but no extracellular antagonist for EGFR in human cells was known. Here, the authors report that the macrophage migration inhibitory factor (MIF), which is a cytokine also implicated in tumorigenesis, binds to the extracellular domain of EGFR and blocks EGF binding, thereby inhibiting EGFR activation and signalling. The authors found that MIF is O-GlcNAcylated at Ser112 and Thr113, and that these modifications are required for EGFR inhibition and for reducing EGF-induced tumour cell invasion in mice. Furthermore, EGFR activation led to the degradation of extracellular MIF, owing to enhanced secretion of matrix metalloproteinase 13. This uncovers a positive feedback loop, by which EGFR signalling can be amplified in heterogeneous tumour microenvironments.