Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Bacillus sp. and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by propan-2-ol and purified to homogeneity by C18 solid phase extraction and reversed phase HPLC. The molecular mass of the purified lactolisterin BU was 5160.94 Da and an internal fragment, AVSWAWQH, as determined by N-terminal sequencing, showed low level similarity with existing antimicrobial peptides. Curing and transformation experiments revealed the presence of a corresponding bacteriocin operon on the smallest plasmid pBU6 (6.2 kb) of strain BGBU1-4. Analysis of the bacteriocin operon revealed a leaderless bacteriocin of 43 amino acids that exhibited similarity to bacteriocin BHT-B (63%), from Streptococcus ratti, a bacteriocin with analogy to aureocin A.IMPORTANCE Lactolisterin BU, broad spectrum leaderless bacteriocin produced by L. lactis subsp. lactis bv. diacetylactis BGBU1-4 strain, expresses strong antimicrobial activity against food spoilage and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Bacillus sp. and streptococci. Lactolisterin BU showed highest similarity with aureocin like bacteriocins produced by different bacteria. Operon for synthesis is plasmid located on the smallest plasmid pBU6 (6.2 kb) of strain BGBU1-4, indicating possible horizontal transfer among producers.