Probiotic developments recently have provided new avenues for the fight against cancer. Via making use of Lactobacillus brevis extract's reducing properties. Therefore, the current study investigated whether nano-exopolysaccharide (NEPS) from probiotic can decrease cell proliferation, increase apoptosis, and activate autophagy in CaCo2 and HT-29 cells using in vitro techniques. On the other hand, compared to non-apoptotic cells, apoptotic cells showed orange-colored bodies as a result of cellular shrinkage and hemorrhage brought on by NEPS stimulation. Not to be overlooked is the NEPS 's exceptional selectivity in causing cell death, particularly in CaCo2 and HT-29 cells. For CaCo2 and HT-29 cells, the half-maximum inhibitory concentration (IC50) values were 9.4 μg/ml and 6.2 μg/ml, respectively. In HT-29 cells, probiotics cause an increase in the synthesis of cytokines linked to inflammation, such as TNF-β, IL-8, and IL-6. As an outcome of apoptosis being triggered and immune system responses being strengthened, our study's findings suggest that using probiotics could be a practical approach to eliminating colorectal malignant cells.