Epidermal growth factor (EGF) and its receptor (EGFR) are involved in many aspects of the development of carcinomas, including tumor cell growth, vascularization, invasiveness, and metastasis. Because EGFR has been found to be overexpressed in many tumors of epithelial origin, it is a potential target for antitumor therapy. Here we report that potato carboxypeptidase inhibitor (PCI), a 39-amino acid protease inhibitor with three disulfide bridges, is an antagonist of human EGF. It competed with EGF for binding to EGFR and inhibited EGFR activation and cell proliferation induced by this growth factor. PCI suppressed the growth of several human pancreatic adenocarcinoma cell lines, both in vitro and in nude mice. PCI has a special disulfide scaffold called a T-knot that is also present in several growth factors including EGF and transforming growth factor alpha. PCI shows structural similarities with these factors, a fact that can explain the antagonistic effect of the former. This is the first reported example of an antagonistic analogue of human EGF.