Activation of epidermal growth factor receptor (EGFR), which occurs in many types of tumour, promotes tumour progression(1,2). However, no extracellular antagonist of human EGFR has been identified. We found that human macrophage migration inhibitory factor (MIF) is O-GlcNAcylated at Ser 112/Thr 113 at its carboxy terminus. The naturally secreted and O-GlcNAcylated MIF binds to EGFR, thereby inhibiting the binding of EGF to EGFR and EGF-induced EGFR activation, phosphorylation of ERK and c-Jun, cell invasion, proliferation and brain tumour formation. Activation of EGFR through mutation or its ligand binding enhances the secretion of MMP13, which degrades extracellular MIF, and results in abrogation of the negative regulation of MIF on EGFR. The finding that EGFR activation downregulates its antagonist in the tumour microenvironment represents an important feedforward mechanism for human tumour cells to enhance EGFR signalling and promote tumorigenesis.