The ADAM10 transmembrane metalloprotease cleaves a variety of cell surface proteins with importance in disease, including ligands for receptor tyrosine kinases of the erbB and Eph families. ADAM10-mediated cleavage of ephrins, the ligands for Eph receptors, is suggested to control Eph/ephrin-mediated cell-cell adhesion and segregation, important during normal developmental processes, and implicated in tumour neo-angiogenesis and metastasis. We previously identified a substrate binding pocket in the ADAM10 C domain that binds the EphA/ephrin-A complex thereby regulating ephrin cleavage. We have now generated monoclonal antibodies specifically recognising this region of ADAM10, which inhibit ephrin cleavage and Eph/ephrin-mediated cell function, including ephrin-induced Eph receptor internalisation, phosphorylation and Eph-mediated cell segregation. Our studies confirm the important role of ADAM10 in cell-cell interactions mediated by both A- and B-type Eph receptors, and suggest antibodies against the ADAM10 substrate-recognition pocket as promising therapeutics, acting by inhibiting cleavage of ephrins and potentially other ADAM10 substrates.