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It is well known that β-conglycinin, a soybean allergen, induces allergies and causes intestinal damage in fetuses and neonates. However, the underlying mechanisms responsible for the adverse effects of β-conglycinin remain elusive. In particular, it is unknown whether or not this dietary substance causes direct damage affecting the proliferation and integrity of intestinal cells. This study evaluated the effect of different concentrations of β-conglycinin (0 to 1,500 µg/mL) and the duration of culture (48 or 72 h) on the proliferation and proteome of porcine intestinal epithelial cells. Eight individually housed piglets (10 d old; initial BW, 3.79 ± 0.07 kg) were randomly divided into 2 groups (n = 4) and challenged with or without β-conglycinin via oral administration d 10 through 28. After the last administration of β-conglycinin or PBS, piglets were killed and jejuna mucosal samples were collected for proteomic analysis. Supplementing β-conglycinin to either culture medium or weanling pigs increased (P < 0.05) the expression of proteins related to apoptosis, stress, and inflammation, but decreased (P < 0.05) the expression of proteins related to cytoskeleton and nucleus replication in intestinal cells. Further analysis confirmed an increase in caspase-3 expression in the cells exposed to β-conglycinin in vivo and in vitro. Collectively, these novel results indicate that β-conglycinin directly induces intestinal damage by depressing intestinal-cell growth, damaging the cytoskeleton, and causing apoptosis in the piglet intestine.