Brachyspira pilosicoli is a gram-negative spiral-shaped, obligate anaerobe bacterium belongs to the spirochete family, the pathogen causing diarrhea, reduced growth rate, reduced egg and meat production, and quality. In recent years, there has been a substantial rise in the rate of Brachyspira pilosicoli infections, as well as the emergence of the virulent and antibiotic-resistant Brachyspira pilosicoli strains. So there is an urgent need for the identification of the therapeutic potential target and development of new drugs for the treatment and prevention of Brachyspira Pilosicoli infections. In the current investigation, we used approach by reference protein (RefSeq. Protein) the individual 1378 protein sequence was entered in DELTA-BLAST against proteomes of pigs (taxid: 9821) pigs & lt; 2 & gt; (taxid: 9823), chickens (taxid: 9031) and result of 27 protein sequences no similarity was found, these 27 protein sequences were again compared against the database of essential single genes individually and 9 of them had no similarity and 18 protein sequences were taken out which had similarity and their protein name, biological process, molecular function and role in virulence have been mentioned and discussed.