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β-Casofensin is a milk-derived bioactive peptide that interacts with intestinal goblet cells. We aimed to determine whether β-casofensin could prevent intestinal dysfunctions induced by neonatal maternal separation (NMS) and whether it retains its protective effects when administered in a casein matrix. We also evaluated whether a casein matrix enriched in β-casofensin protects against intestinal enteritis induced by indomethacin. β-Casofensin abolished NMS-induced jejunal hyperpermeability and prevented the depletion of goblet and Paneth cells induced by NMS. In addition, β-casofensin maintained its effectiveness against NMS-induced intestinal barrier alterations when administered in a casein matrix. A casein matrix containing β-casofensin also reduced intestinal damages induced by indomethacin. A functional food containing β-casofensin may prevent both the deleterious effects of neonatal stress on the intestinal barrier and indomethacin-induced enteritis. These results suggest a promising application of β-casofensin as a gut barrier protector in the context of bioactive foods and clinical nutrition.