Probiotic Pediococcus pentosaceus GS4 produces conjugated linoleic acid (CLA) through biohydrogenation, which may be vital for colon cancer mitigation. Here, we have appraised the anti-cancer potential of probiotic GS4 against colon cancer. CLA produced by probiotic GS4 reduced the proliferation of HCT-116 in vitro rendering towards apoptosis and subsequently abrogated NF-κB and p-Akt. Probiotic GS4 efficiently mitigated colon cancer in azoxymethane (AOM)-induced colon cancer mice model, which was evident from reduced disease severity, alleviated oxidative stress and neoplastic characters. Further delineation of underlying consequences at the level of host-microbe interface, the probiotic GS4 arbitrated the gut microflora-associated biohydrogenation, which subsequently triggered apoptosis in colonocytes, apparent from PARP cleavage, caspase 3 activity, DNA fragmentation and inhibition of HDAC activity. Conclusively, modulation of host-microbe interface with selective colonizing strategies combined with unique biohydrogenation ability of probiotic GS4 promises to be a therapeutic/functional food supplement responsible for mitigation of colon cancer. © 2016 Elsevier Ltd.