Food allergy has become a highly prevalent immune-related disease worldwide, and intestinal barrier damage is a critical step in sensitization to food allergens. Silkworm pupae are widely consumed as a high-quality protein source; however, the intestinal effects of Bomb m 6, a major novel lipoprotein allergen from silkworm pupae, remain unclear, which limits the prevention and control of silkworm pupa allergy and the safe utilization of silkworm resources. In this study, Caco-2 cells, HT-29 cells, and a Caco-2/HT-29 co-cultured intestinal barrier model were used to investigate the effects of Bomb m 6 on intestinal epithelial cell viability, inflammatory cytokines, alarmins, and barrier function using CCK-8 assay, ELISA, FITC-Dextran permeability assay, and Western blot. The results showed that 6.25–200 μg·mL?¹ Bomb m 6 had no significant cytotoxicity toward Caco-2 cells, whereas 400 μg·mL?¹ Bomb m 6 significantly inhibited the viability of HT-29 cells. Treatment with 100 and 200 μg·mL?¹ Bomb m 6 increased IL-6 and IL-8 levels in Caco-2 cells to 6.80 and 31.20 pg·mL?¹, respectively. Treatment with 200 μg·mL?¹ Bomb m 6 significantly increased the levels of IL-33, IL-25, and TSLP in Caco-2 cells, while concentrations of 50 μg·mL?¹ and above significantly increased IL-1β, TNF-α, IL-33, and TSLP levels in HT-29 cells. After treatment with 200 μg·mL?¹ Bomb m 6 for 24 h, the apparent permeability coefficient (Papp) of the in vitro intestinal barrier model increased by 2.5-fold, IL-6 and IL-8 secretion was enhanced, and the expression levels of the tight junction proteins ZO-1, Occludin, and Claudin-1 were significantly downregulated. These results suggest that Bomb m 6 may impair intestinal epithelial barrier function by inducing inflammatory responses in intestinal epithelial cells, reducing tight junction protein expression, and increasing barrier permeability, thereby providing experimental evidence for elucidating the mechanism of silkworm pupa allergy, its prevention and diagnosis, and the safe development of silkworm resources.