Dysfunction of the intestinal epithelial barrier plays an important role in the pathogenesis of several intestinal diseases, including celiac disease, inflammatory bowel disease, and irritable bowel syndrome. The present research was carried out to investigate the protective effect of total polysaccharides of adlay bran (TPA) on TNF-α-evoked epithelial barrier dysfunction in Caco-2 cells. Caco-2 cells were treated with or without TPA in the absence or presence of TNF-α, and transepithelial electrical resistance (TEER) and Phenol Red flux were assayed to evaluate the intestinal epithelial barrier function. The results indicated that TPA suppressed the TNF-α-induced release of pro-inflammatory factors. Furthermore, TPA obviously assuaged both the increased paracellular permeability and the decrease of TEER in TNF-α-challenged Caco-2 cells. Furthermore, TPA obviously assuaged TNF-α-evoked up-regulation of IL-8 and IL-6 expression, down-regulation of occludin and ZO-3 expression, and markedly suppressed the activation and protein expression of NF-κB p65. Our results indicated that TPA assuages the TNF-α-evoked dysfunction of the intestinal epithelial barrier by inhibiting the NF-κB p65-mediated inflammatory response.