Lactobacillus rhamnosus CICC 20975 produces a 6,502-Da bacteriocin, named bacteriocin RC 20975, active against Alicyclobacillus acidoterrestris, Lactobacillus acidophilus, Lactobacillus brevis, Bacillus subtilis, and Listeria innocua. This bacteriocin is not quite heat stable but is effective after refrigerated storage and freeze-thaw cycles. Bacteriocin RC 20975 was added at a concentration of 256 AU/ml to the endospores of A. acidoterrestris DSM 3922; no viable cells were detected after 24 h. The primary mode of action of bacteriocin RC 20975 seems to be the formation of pores, as indicated by K⁺ efflux from metabolically active cells of A. acidoterrestris. However, efflux of larger cytoplasmic content was not observed within the first 30 min after bacteriocin RC 20975 treatment. In addition, adsorption of bacteriocin RC 20975 to target cells at different temperatures and pH levels and in the presence of surfactants was studied. Finally, the effect that different media, media components, and addition of vitamins to the media had on bacteriocin RC 20975 production was also studied.