Microcin B17 (McB) is a 43-amino acid antibacterial peptide targeting the DNA gyrase. The McB precursor is ribosomally produced and then post-translationally modified by the Mcb-BCD synthase. Active mature McB contains eight oxazole and thiazole heterocycles. Here, we show that a major portion of mature McB contains an additional unusual modification, a backbone ester bond connecting McB residues 51 and 52. The modification results from an N → O shift of the Ser 52 residue located immediately downstream of one of McB thiazole heterocycles. We speculate that the N,O-peptidyl shift undergone by Ser 52 is an intermediate of post-translational modification reactions catalyzed by the McbBCD synthase that normally lead to formation of McB heterocycles.