Microcin J25 is a ribosomally synthesised 21-residue antimicrobial peptide produced by certain strains of enterobacteria, that adopts an extraordinary 'threaded lasso' structure. To date, the biosynthesis of this peptide is little understood. Here we report the in vitro maturation of the microcin precursor peptide into active microcin J25 for the first time. Furthermore, we show that the enzymes required for the posttranslational modification of this precursor peptide are associated with the bacterial inner membrane.