This study aimed to investigate the effect of milk peptides on irritable bowel syndrome (IBS). The mice were intragastrally administered with casein or whey protein hydrolysates at a dose of 1 g/kg body weight/day for 24 days and were subjected to Citrobacter rodentium infection and water avoidance stress from day 7 to 24. Results indicated that casein and whey protein hydrolysates effectively reduced diarrhea, anxiety, and visceral hypersensitivity in IBS mice. Casein and whey protein hydrolysates regulated gut microbiota composition and increased the abundance of short-chain fatty acid-producing bacteria, such as Alloprevotella and Alistipes. Whey protein hydrolysate significantly increased the mRNA levels of zonula occludens-1 (ZO-1) and claudin-1 in the colon, while casein hydrolysate significantly improved the mRNA levels of occludin. Casein and whey protein hydrolysates both decreased the levels of pro-inflammatory cytokines including interleukin-6 (IL-6), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha), while increased the level of anti-inflammatory cytokine interleukin-10 (IL-10). Importantly, casein and whey protein hydrolysates significantly reduced colonic mast cell activation and decreased prostaglandin E2 (PGE2) production. Moreover, three novel casein-derived cyclooxygenase-2 (COX2)-inhibitory peptides RGPF, FPK, and NPW were identified with IC(50) values of 0.36 +/- 0.03, 0.64 +/- 0.01, and 1.10 +/- 0.09 mM, respectively and predicted to form hydrogen bonds and hydrophobic interactions with the residues of the active site of COX2. This study highlighted the potential of milk peptides as bioactive ingredients in functional foods for managing IBS.