A major driving force for human cancers is the dysregulation of epidermal growth factor signalling, which promotes cell survival, proliferation and migration. Cell surface proteases play a crucial role in this process by releasing ligands which activate the epidermal growth factor receptor (EGFR). In Drosophila, EGFR ligands are cleaved, secreted and thus activated by the rhomboid intramembrane proteases. In humans, however, EGFR ligand activation is known to be catalysed mainly by ADAM metalloproteases. Whilst rhomboids are conserved in humans, their role is poorly defined because of the limited number of known substrates. The human rhomboid protease RHBDL2 is highly expressed in epithelial tissues and is localised to the plasma membrane. Using a quantitative proteomics approach in human keratinocytes we reveal a number of novel substrates of RHBDL2 and surprisingly find EGFR to be the major substrate. Keratinocytes lacking RHBDL2 activity exhibit substantially increased collective cell migration. This can be inhibited by pharmacological suppression of the EGFR kinase or ADAM protease activity, re-introduction of RHBDL2 activity, addition of conditioned medium from cells expressing RHBDL2, or addition of recombinant extracellular domain of EGFR. In summary, these data show that RHBDL2 suppresses autocrine activation of EGFR in epithelial cells by shedding the EGFR ectodomain which intercepts free endogenous EGFR ligands and thus lowers their pericellular concentration. These data imply that rhomboid proteases may be involved in the fine-tuning of EGFR signalling in mammals more than currently appreciated.