Aims: Purification and characterization of a novel bacteriocin produced by strain Enterococcus faecium D081821. Methods and Results: Enterococcus faecium D081821, isolated from the traditional Taiwanese fermented food dochi (fermented black beans), was previously found to produce a bacteriocin against Listeria monocytogenes and some Gram-positive bacteria. This bacteriocin, termed enterocin TW21, was purified from culture supernatant by ammonium sulfate precipitation, Sep-Pak C-18 cartridge, ion-exchange and gel filtration chromatography. Mass spectrometry analysis showed the mass of the peptide to be approximately 5300.6 Da. The N-terminal amino acid sequencing yielded a partial sequence NH2-ATYYGNGVYxNTQK by Edman degradation, and it contains the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The open reading frame (ORF) encoding the bacteriocin was identified from the draft genome sequence of Enterococcus faecium D081821, and sequence analysis of this peptide indicated that enterocin TW21 is a novel bacteriocin. Conclusions: Enterococcus faecium D081821 produced a bacteriocin named enterocin TW21, the molecular weight and amino acid sequence both revealed it to be a novel bacteriocin. Significant and Impact of Study: A new member of class IIa bacteriocin was identified. This bacteriocin shows great inhibitory ability against L.monocytogenes and could be applied as a natural food preservative.