Abstract: The purpose of this study was to establish an endoplasmic reticulum stress model induced by β-conglycinin using intestinal epithelial cells from hybrid yellow catfish. The method involved isolating intestinal epithelial cells using an enzyme digestion method. Cells were identified through morphological assessment, cytokeratin-18 (CK-18) immunofluorescence, and alkaline phosphatase staining. Following isolation, well-grown cells were stimulated with different levels of β-conglycinin for 24h. RT-qPCR was employed to measure mRNA expression of relative indexes to determine the optimal stimulation concentration. Subsequently, cells were stimulated at the optimal concentration for 0, 12h, 24h and 36h. RT-qPCR, CCK8, transmission electron microscopy, and immunofluorescence were used for measuring the relative indexes to determine the suitable stimulation time. The Results indicated that: (1) Successful isolation of intestinal epithelial cells from hybrid yellow catfish, characterized by paving stone-like cell morphology and positive CK-18 and alkaline phosphatase staining. (2) Identification of 4 mg/mL as the optimal stimulation concentration. Treatment with 4 mg/mL β-conglycinin significantly elevated mRNA levels of ERS-, autophagy-, apoptosis- and inflammation related genes (P<0.05), except for grp78, jnk, il-10 and atf6 genes. (3) Determination of 24h as the optimal stimulation duration. Cell viability significantly decreased with prolonged stimulation time. Notably, after 24h of treatment, swellen endoplasmic reticulum, elevated levels of GRP78, LC3, Caspase3 protein, Tunle signal, and the mRNA expression of perk, atf6, beclin1, lc3a, bcl2, caspase3, caspase9 and il-12 were observed (P<0.01). In conclusion, this study successfully isolated intestinal epithelial cells from hybrid yellow catfish and established the endoplasmic reticulum stress model. These findings provides a foundation for understanding the effects of unfolded protein response on soybean meal-induced enteritis of hybrid yellow catfish.