Lactiplantibacillus plantarum B21 isolated from Vietnamese sausage (nem chua) has previously displayed broad antimicrobial activity against Gram-positive bacteria including foodborne pathogens Listeria monocytogenes and Clostridium perfringens. This study successfully identified the antimicrobial agent as plantacyclin B21AG, a 5668 Da circular bacteriocin demonstrating high thermostability, resistance to a wide range of pH, proteolytic resistance and temporal stability. We report a reverse genetics approach to identify and characterise plantacyclin B21AG from first principles. The bacteriocin was purified from culture supernatant by a three-step process consisting of concentration, n-butanol extraction and cation exchange chromatography. A de novo peptide sequencing using LC-MS/MS techniques identified two putative peptide fragments which were mapped to the genome sequence of L. plantarum B21. This revealed an ORF corresponding to a putative circular bacteriocin with a 33-amino acid leader peptide and a 58-amino acid mature peptide encoded on a native plasmid pB21AG01. The bacteriocin is shown to be a small cationic predominantly α-helical protein (69%). The corresponding gene cluster, consisted of seven genes associated with post-translational circularisation, immunity and secretion. Whilst plantacyclin B21AG is 86% identical to the newly published plantaricyclin A it is more highly cationic having a net charge of +3 due to an additional basic residue in the putative membrane interaction region. This and other substitutions may well go some way to explaining functional differences. The robust nature of plantacyclin B21AG, its antimicrobial activity and associated machinery for cyclisation make it an interesting biotechnological target for development, both as a food-safe antimicrobial or potentially a platform technology for recombinant protein circularisation.