Aims To purify and primarily characterize an anti-Alicyclobacillus bacteriocin produced by Bifidobacterium animalis subsp. animalis CICC 6165, suggested to be named bificin C6165. Methods and Results During purification of the bificin C6165, optimal recovery was achieved with ammonium sulfate precipitation followed by two chromatographic steps. Mass spectrometry analyses revealed a distinctive peak corresponding to a molecular mass of 3395 center dot 1Da. This bacteriocin was heat stable, effective after refrigerated storage and freezethaw cycles. The primary mode of action of bificin C6165 is most probably due to pore formation, as indicated by the efflux of K+ from metabolically active cells of Alicyclobacillus acidoterrestris. In the presence of 10mmoll1 gadolinium, bificin C6165 did not affect cells of Alicyclobacillus acidoterrestris. This suggests that the mode of action of bificin C6165 relies on a net negatively charged cell surface. Conclusions Bificin C6165 is indeed a novel bacteriocin and it exhibited remarkable potency for Alicyclobacillus control. Significance and Impact of the Study Application of bacteriocins in preservation of fruit juices has seldom been studied. Bificin C6165 may be an alternative method to control juice spoilage by this Alicyclobacillus acidoterrestris and meet increasing consumer demand for nature and artificial chemical additive-free food products.