<jats:p>Microcin J25 (MccJ25) is the single representative of the immunity group J of the microcin group of peptide antibiotics produced by Enterobacteriaceae. It induces bacterial filamentation in susceptible cells in a non‐SOS‐dependent pathway [R. A. Salomon and R. Farias (1992) <jats:italic>J. Bacteriol.</jats:italic><jats:bold>174</jats:bold>, 7428–7435]. MccJ25 was purified to homogeneity from the growth medium of a microcin‐overproducing <jats:italic>Escherichia coli</jats:italic> strain by reverse‐phase HPLC. Based on amino acid composition and absolute configuration determination, liquid secondary ion and electrospray mass spectrometry, extensive two‐dimensional NMR, enzymatic and chemical degradations studies, the structure of MccJ25 was elucidated as a 21‐residue peptide, <jats:italic>cyclo</jats:italic>(‐Val<jats:sup>1</jats:sup>‐Gly‐Ile‐Gly‐Thr‐Pro‐Ile‐Ser‐Phe‐Tyr‐Gly‐Gly‐Gly‐Ala‐Gly‐His‐Val‐Pro‐Glu‐Tyr‐Phe<jats:sup>21</jats:sup>‐). Although MccJ25 showed high resistance to most of endoproteases, linearization by thermolysin occurred from cleavage at the Phe<jats:sup>21</jats:sup>‐Val<jats:sup>1</jats:sup> bond and led to a single peptide, MccJ25‐L. While MccJ25 exhibited remarkable antibiotic activity towards <jats:italic>Salmonella newport</jats:italic> and several <jats:italic>E. coli</jats:italic> strains (minimal inhibitory concentrations ranging between 0.01 and 0.2 µg·mL<jats:sup>–1</jats:sup>), the thermolysin‐linearized microcin showed a dramatic decrease of the activity, indicating that the cyclic structure is essential for the MccJ25 biological properties. As MccJ25 is ribosomally synthesized as a larger peptide precursor endowed with an N‐terminal extremity, the present study shows that removal of this extension and head–tail cyclization of the resulting propeptide are the only post‐translational modifications involved in the maturation of MccJ25, that appears as the first cyclic microcin.