INTRODUCTION The rapid increase and spread of drug resistance pathogens have constrained the thought in scientists to exchange strategies for battling contaminations and infections. One big limitation of utilizing wide spectrum antibacterial agents is that these almost kill any type of bacteria which do not particularly resist a drug. The drugs which kill a wide range of bacteria create drug resistance in both pathogenic bacteria and commensals microorganisms (Walker and Levy, 2001). Antimicrobial substances produced by microorganisms have changed the microbial ecology. Bacteriocins are secondary metabolites and belong to class of antimicrobial substances produced by a variety of bacteria. They are robisomally- synthesized proteins or peptides and have antibacterial action (Gálvez et al.,2007). Bacteriocins show antimicrobial action often against closely associated bacterial species (Cladera‐ Olivera et al., 2004). Bacteriocins have great variation even within same species of bacteria (Motta and Brandelli, 2008). Bacteriocins are also defined as biologically active peptides with bactericidal mode of action. They include a variety of protein molecules having different size, activity, immune mechanisms as well as different antimicrobial targets (Heng et al., 2007). As compared to traditional antibiotic compounds, they are different from them in one special way that they are narrow spectrum and kill only those bacteria which are closely related to each other (Riley and Wertz,2002). Most of the species from family Bacillus are likewise considered as GRAS bacteria and they are critical bacteriocin producers (Martirani et al., 2002). Bacillus group bacteria are known for the production of various antimicrobial substances like peptides, lipopeptides, antibiotics, and as well as bacteriocins. There many species of Bacillus genus that have ability to produce a variety of useful antimicrobial products like bacteriocins and among them B. subtilis is the most important one. This species is considered nonpathogenic and non-toxic to humans as well as plants to cause disease (Stein, 2005). The bacteriocins produced by one species of bacteria can be applied as antibacterial against other related bacteria. For example, bacteriocins produced by B. subtilis have been proved experimentally to be used against Listeria monocytogenes infection (Sabaté and Audisio, 2013).Almost 90% bacteria have ability to produce bacteriocin products and many of these have broad spectrum inhibitory activity for different microbes (Bizani and Brandelli, 2002).The current research was performed to check potential of B. subtilis bacterium for bacteriocin production isolated from soil samples and its toxicity evaluation in experimental animals. Pak. J. Agri. Sci., Vol. 57(5), 1403-1411; 2020 ISSN (Print) 0552-9034, ISSN (Online) 2076-0906 DOI: 10.21162/PAKJAS/20.10026 http://www.pakjas.com.pk PRODUCTION AND THERAPEUTIC POTENTIAL OF BACTERIOCIN PRODUCED BY INDIGENOUS ISOLATES OF Bacillus subtilis Riffat Shamim Aslam 1 , Muhammad Ashraf 1,* , Mashkoor Mohsin 1 and Zafar Iqbal 2 1 Instititute of Microbiology, Faculty of Veterinary Science, University of Agriculture Faisalabad, Punjab, Pakistan; 2 Department of Parasitology, Faculty of Veterinary Science, University of Agriculture Faisalabad, Punjab, Pakistan *Correspondence author’s e-mail: mashraf@uaf.edu.pk Bacteriocins are the antimicrobial peptides that are produced by many of the bacterial species as secondary metabolites. They have potential to inhibit the growth of many other pathogenic bacteria. The present study was conducted to check production and therapeutic potential of bacteriocin produced by different indigenous isolates of Bacillus subtilis. Soil samples were collected from different environments including soil mud, agricultural land soil, sewage, and deteriorating plant matter. These samples were cultured on Nutrient Agar for isolation of B. subtilis. These isolates were further purified by culturing and identified by biochemical.16SrDNA technique was used to confirm the B. subtilis. The confirmed isolates were evaluated for their potential to produce bacteriocin using agar well diffusion method. Bacteriocin was purified using Ammonium sulfate precipitation method, and its chemotherapeutic properties were checked by developing two models, Superficial skin model and experimental Gram negative septicemia model. The results indicated that B. subtilis isolates showed a band of 1311bp on gel in 16SrDNA analysis and sequencing also confirmed B. subtilis isolates. No toxic effect of bacteriocin was observed in experimental animals. There was no mortality in treated and controlled group animals during experimentation. In conclusion, bacteriocin peptides have ability to inhibit bacterial growth and may be exploited to treat microbial infections. Keywords: Bacteriocin, Secondary metabolites, Toxic effect, 16SrDNA, Indigenous.