Aims: This study aimed to investigate the effect of exopolysaccharide (EPS) from Bifidobacterium pseudocatenulatum ATCC 27919 in Caco-2 cells on apoptotic and autophagic pathways. Methodology and results: Cell viability was examined by MTS assay and it showed a significant decrease in Caco-2 cells after exposure to EPS. Microscopy imaging and morphological analyses demonstrated that EPS-exposed Caco-2 cells exhibited the main morphological characteristics of apoptosis. EPS-exposed cells showed early apoptosis and cell cycle arrest at the G2/M phase in the cell cycle and dead cell assays. qPCR suggested that mRNA expression of substantial apoptotic markers such as cleaved Caspase-3, BAX and PARP-1 were significantly increased in EPS-exposed cells. The autophagy event was demonstrated in EPS-exposed cells by the contrary mRNA expression of Beclin-1 and Bcl-2 and the detection of autophagic LC3-II protein at 24 h exposure. GRP78 mRNA expression was also increased in EPS-exposed cells, indicating the occurrence of endoplasmic reticulum (ER) stress. Conclusion, significance and impact of study Autophagy activity in EPS-exposed Caco-2 cells preceded apoptosis, suggesting it was a cytoprotective response against ER stress. Research findings set the foundation for therapeutic CRC treatments and provide insight into its regulatory processes.