Probiotic treatment has been gaining attention due to its remarkable effects in alleviating toxicity and carcinogenesis. The novel strain Pediococcus pentosaceus GS4 has been reported for probiotic, survivability in simulated gastrointestinal fluid, and antioxidative and biohydrogenation properties. Therefore, we hypothesize that this specific strain might be able to assuage the effect of azoxymethane (AOM)-induced toxicity in mice. Twenty-eight Swiss albino mice were divided into 4 groups and were studied for 32 weeks. Azoxymethane (10 mg/kg body weight) was administered intraperitoneally twice (0th and 14th days), and probiotic GS4 (1.1 × 10(9) colony-forming unit/mL) was given orally for the respective groups. Mice who served as the normal control received only normal saline. GS4-intervened AOM-induced mice showed marked improvement at the histopathologic level, in the liver and kidney. Moreover, probiotic GS4 intervention in AOM-induced mice exhibited a significant reduction in the liver function biomarker when compared with the AOM-induced mice. Probiotic GS4 intervention reduced the intestinal structural deformities as evident from the elevated brush border membrane-associated disaccharidases (sucrase, lactase) and intestinal alkaline phosphatase activities, which were found disrupted by AOM intoxication. Fecal bacterial load was found to be reduced in AOM-induced mice which were subsequently replenished by the probiotic GS4 intervention as apparent from the enhanced fecal bacterial load. There were no adverse effects observed in the probiotic control group. Conclusively, novel probiotic strain GS4 exhibited safe and beneficial effects against the toxicity threats posed by AOM. Thus, GS4 could be considered as a potential food supplement/additive for therapeutic purposes in gastrointestinal disorders related to inflammation and cancer.