Background:Necrotic enteritis is a widespread disease in poultry caused by Clostridium perfringens. We previously reported that dietary arginine supplementation protected the intestinal mucosa of broiler chickens with necroticenteritis, but the related protective mechanisms remain unclear. The in vivo trial was designed as a 2 × 2 factorial arrangement to evaluated the effects of arginine supplementation on inflammatory responses, arginine transporters, arginine catabolism and JAK-STAT signalling pathway in broiler chickens challenged with C. perfringens or without C. perfringens. Furthermore, we validated the in vivo results using intestinal epithelial cells of chicken embryos. Results:C. perfringens infection markedly increased gut gross pathological and histopathological lesion scores, promoted liver C. perfringens invasion, reduced serum arginine levels, and elevated jejunal mucosal lysozyme activities (P<0.05), but these effects were significantly reversed by arginine supplementation in vivo (P<0.05). The challenge significantly increased serum procalcitonin levels, jejunal mucosal iNOS activities and jejunal IL-6, TGF-β3, cationic amino acid transporter (CAT)-1, and CAT-3 mRNA expression (P<0.05), whereas arginine supplementation significantly reduced jejunal IFN-γ, IL-1β, IL-6, IL-10, TGF-β3, and CAT-3 mRNA expression (P<0.05). Arginine supplementation significantly attenuated the C. perfringens challenge-induced increases in jejunal iNOS, arginase 2, arginine decarboxylase, arginine:glycine amidinotransferase, JAK1, JAK3, STAT1, and STAT6 mRNA expression (P<0.05). The in vitro experiment showed that C. perfringens challenge markedly increased cellular cytotoxicity and the mRNA expression of IL-1β, IL-8, IL-10, CAT-1 and CAT-3 (P<0.05), which were significantly reversed by 50μmol/L and/or 400μmol/L arginine pre-treatment (P<0.05). Conclusions:Arginine prevented C. perfringens challenge-induced circulated arginine deficiency, normalized intestinal arginine transport and catabolism, down-regulated JAK-STAT signalling pathway and attenuated the inflammatory response, which exerted protective effects on the intestine of broiler chickens.