Probiotics with ability to produce conjugated linoleic acid (CLA) is considered as an additive health benefit property for its known role in colon cancer mitigation. The conversion involves the biohydrogenation of the unsaturated fatty acid into conjugated form. Probiotic strain Pediococcus spp. GS4 was efficiently able to biohydrogenate linoleic acid (LA) into its conjugated form within 48 h of incubation. Quantum of CLA produced with a concentration of 121 μg/ml and sustained cell viability of 8.94 log cfu/ml maximally. Moreover, antibacterial effect of LA on the strain ability for biohydrogenation was examined at different concentrations and concluded to have a direct relationship between LA and amount of CLA produced. The efficiency of the strain for CLA production at different pH was also estimated and found maximum at pH 6.0 with 149 μg/ml while this ability was reduced at pH 9.0 to 63 μg/ml. Sesame oil, which is rich in the triacylglycerol form of LA, was also found to act as a substrate for CLA production by Pediococcus spp. GS4 with the aid of lipase-catalyzed triacylglycerol hydrolysis and amount of CLA produced was 31 μg/ml at 0.2 % while 150 μg/ml at 1.0 % of lipolysed oil in skim milk medium. Conjugated form was analyzed using UV scanning, RP-HPLC, and GC-MS. This study also focused on the alternative use of lipolysed sesame oil instead of costly LA for biohydrogenation and could be a potential source for the industrial production of CLA.