Lactic acid bacteria (LAB) are economically important because of their fermentative aptitude and they also confer healthand nutritional benefits. Pediococcus acidilactici NCDC 252 (NCDC 252) is potential probiotic with novel genome thatexhibited all in-vitro probiotic attributes, colonization in intestine and survival in conditions that mimick gastric, intestinaland vaginal fluid. Genome was screened for its ability to produce antimicrobial peptide (AMP). AMP from NCDC 252 wasisolated and purified against indicator strain (Escherichia coli). AMP production was optimized in MRS broth at 32 °C, pH6 and upto 36 h of incubation under both aerobic and anaerobic conditions. AMP was purified 2.2 fold with yield of 7.2%and specific activity of 64.5 U mg/mL using ammonium sulphate precipitation (upto 70%) and gel filtration chromatography.Molecular weight of purified AMP was calculated to be 2.5 KDa. Tricine SDS-PAGE and LC/MS studies confirmed it to bea low molecular weight peptide. AMP was stable over pH range of 3 to 9 and temperature range of 40 °C to 100 °C. Enzymatictreatment inactivated the AMP whereas, detergent treatment enhanced its activity.