ABSTRACT: Fermentation of raw marine food is popular in Japan, but is occasionally associated with Clostridium botulinum outbreaks. Lactic acid bacteria in fermented food produce antibacterial substances (ABS). An ABS producing strain Iz.3.13 was isolated from commercially fermented ‘izushi’. The purpose of this study was to characterize strain Iz.3.13 and the ABS it produced. Strain Iz.3.13 was identified as Pediococcus pentosaceus by morphological, biochemical characteristics and 16S rDNA sequence similarity. Strain Iz.3.13ABS produced and ABS inhibited Listeria and Cl. botulinum. The ABS was inhibited by α-chymotrypsin and proteinase K, but not by catalase, lipase, or α-amylase indicating the ABS was a bacteriocin. The bacteriocin remained active at 100°C for 15 min, and pH 2–8. Mode of action of the bacteriocin against Listeria monocytogenes was bactericidal and bacteriolytic. Partial analysis of the purified bacteriocin by Edman degradation, showed a 22-amino acid residue closely related to Pediocin AcH. However, MALDI-TOF-MS analysis estimated a molecular mass of 4621.6 Da. This is a first report of a Cl. botulinum-inhibiting bacteriocin-producer from traditional Japanese fermented marine food. The bacteriocin produced by Ped. pentosaceus Iz.3.13 might have potential to ensure the safety of fermented marine food.