Microcins are gene-encoded antimicrobial (poly)peptides secreted by Enterobacteriaceae. Produced under conditions of nutrient depletion, they are active against phylogenetically related microbial strains. Therefore, they are considered to play an important role in the microbial competitions within the intestinal flora. Among the limited sample of nine microcins hitherto described, a wide variety of structures and modes of action could be identified. Microcin J25 (MccJ25) is a unique 21-residue macrocyclic peptide recently isolated. It shows highly potent activity on various Salmonella and Escherichia strains (CMI: 1 to 5 nM). MccJ25 three-dimensional structure is highly compact and consists in a twisted antiparallel beta-sheet connecting two loops. The peptide structure, stabilized by hydrogen bonds and hydrophobic interactions only, as well as the antimicrobial activity were shown to be largely maintained upon peptide linearization and denaturing treatments. Such stability is believed to play an essential role in the environmental context of MccJ25 expression. The highly potent activity and narrow spectrum of activity of MccJ25 are in favor of a receptor-mediated mechanism of action. In early studies, bacteria resistant to MccJ25 were reported to display mutations in the gene encoding the outer membrane protein FhuA. FhuA is a high-affinity receptor involved in iron transport. Found in both Salmonella and Escherichia species, it is also used by the T1, T5 and phi80 phages to infect bacteria (cell adhesion and DNA ejection). In this study, we demonstrated that MccJ25 is able to interfere with FhuA biological functions both in vivo and in vitro. MccJ25 was shown to prevent the infection of E. coli by the T5 phage by inhibiting the adhesion step, which strongly suggests that MccJ25 blocks the interaction between the phage and its membrane receptor. This was confirmed in vitro, in the absence of other components of the bacterial membrane, where MccJ25 was also able to inhibit the DNA ejection induced by the irreversible binding of the T5 phage to FhuA. As reported for the iron/ferrichrome complex, the natural ligand of FhuA, the inhibition observed was in favor of a non-competitive mechanism. Finally, direct interaction of MccJ25 with FhuA was shown by size-exclusion chromatography. Therefore we propose that FhuA is the receptor of MccJ25 at the bacterial membrane.