Microcins are gene-encoded antimicrobial peptides secreted by Enterobacteriaceae. Microcin J25 (MccJ25) is a unique 21-residue lasso-type peptide presenting a side chain to backbone cyclization between Glu8 and the N-terminus. The embedded ring is threaded by the C-terminal tail of the molecule, thus forming a noose-like feature. The highly potent activity and narrow spectrum of activity of MccJ25 (CMI ranging from 1 to 600 nM against enterobacterial strains) argue for a receptor-mediated mechanism of action. In early studies, bacteria resistant to MccJ25 were reported to display mutations in the multifunctional outer membrane protein FhuA, the high-affinity transporter of iron-ferrichrome and the receptor for phages T5, T1 and phi80. In this study, we have demonstrated by size-exclusion chromatography that MccJ25 binds directly to FhuA. This interaction was shown to prevent binding of phage T5 to its receptor and subsequently inhibit the ejection of the phage DNA which normally occurs upon binding of the phage to FhuA. We compared MccJ25 to the thermolysin-cleaved variant t-MccJ25 in which the 8-18 beta-hairpin region is disrupted. From our results, thermolysin cleavage both results in a 100 fold dropped antibacterial activity of the peptide and induces a loss of interaction between MccJ25 and FhuA. Therefore we propose that FhuA is the receptor of MccJ25 at the bacterial outer membrane and that the interaction implicates the 8 to 18 residues