Three major enzyme-hydrolyzed peptides have been produced after simulative digestion in vitro of soybean β-conglycinin. The intestinal barrier of IPEC-J2 induced by β-conglycinin enzyme-hydrolyzed peptides was evaluated in this study. The increased alkaline phosphatase (AP) activity was actually linearly correlated with the incubation time by the hydrolysate, the purified 52 kD peptide, or the mixture of 25 and 30 kD peptides. The MTT and TEER values declined in dose-dependence (0–2 mg/mL, p \lt 0.05) or in time-dependence (2–24 h, p \lt 0.05). After treatment with different hydrolyzed peptides, the tight junction expression of claudin-3, claudin-4, occludin, and ZO-1 were reduced (p \lt 0.05). Finally, it is found out that the maximum damage to the epithelial barrier function was induced by the mixture of 25 and 30 kD peptide, whereas the minimum damage was caused by the 52 kD peptide.