A bacteriocin producing lactic acid bacterium was isolated from the fermented cow milk of Kinnaur region of Himachal Pradesh and identified asLactobacillus pentosus RL-2e on the basis of morphological, biochemical and 16S rDNA analysis. L. pentosus exhibited strong inhibitory potential against food spoilage microorganisms. Maximum bacteriocin production was observed in Elliker broth at 30O C, pH 6.5 and 2% inoculum size. The bacteriocin was purified to 6.9 fold with 3% yield using ammonium sulphate precipitation and gel permeation chromatography. The SDS PAGE analysis revealed that the molecular weight of this bacteriocin was around 20 kDa. The study showed that bacteriocin ofL. pentosus has good potential as a food preservative.