Objective: Rumen is rich source of anaerobic microbes and these microbes has ability to produce bacteriocin like substances,The present study was aimed at Partial Purification of bacteriocin like substance from Rumen Liquor obtained from the slaughtered cattle. Methods: Rumen liquor samples were collected from slaughtered cattle of fifteen samples collected the anti- microbial activity was seen only in those animals which were fed just before slaughter. The samples were precipitated by 60% ammonium sulphate saturation these precipitates were further purified using ion-exchange chromatography (SP-Sepharose) and eluted with 1.0 M sodium chloride as a single peak. The purity of protein was confirmed by SDS-PAGE analysis. Results: Antimicrobial activity was obsereved in at 60% ammonium sulphate saturation and these The protein with anti-microbial activity was eluted with 1.0 M sodium chloride as a single peak using ion exchange chromatography, molecular size of these protein was around 6.5kDa,. Further the purified protein was subjected for in gel tryptic digestion and nano LC-MS analysis of the peptides released followed by the identification of protein using MASCOT analysis which had shown similarity with the fimbrial protein of Pseudomonas aeruginosa (SO4440) and oligopeptide ABC transporter, ATP binding protein (B72300)) [23]. Bacteriocin (BViA) falls within the superfamily of ABC transporters and ATP binding proteins demonstrate highest homology matches with the BViA. Conclusion: Thus the observations made in this study clearly demonstrate that the peptide purified from rumen liquor was indeed bacteriocin like substance.