The purpose of this study is to obtain effective and stable bacteriocin-like substance from lactic acid bacteria. Lactobacillus buchneri KLDS1.0364, which was isolated from fermented cream, a traditional dairy product in Inner Mongolia, could produce bacteriocin-like substance. The bacteriocin-like substance produced by KLDS1.0364 was partially purified and the characteristics were studied. The bacteriocin-like substance was purified by SP-Sepharose fast flow cation exchange chromatography. The molecular weight of the bacteriocin-like substance was 21.6kD, as determined by tricine-SDS-PAGE. The bacteriocin-like substance remained stable after 30 min at 121℃ and after 2 h of incubation during pH 2~10. Complete inactivation of antimicrobial activity was observed after treatment of the bacteriocin-like substance with several proteinases. Treatment with catalase and α-amylase did not result in any changes of antimicrobial activity. The mode of action of the bacteriocin-like substance is bactericidal. It exhibited a broad spectrum of antagonistic activity against various species of Gram-positive bacteria, Gram-negative bacteria and fungi.