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Abstract To address the overuse of antimicrobials in poultry production, new functional feed ingredients, i.e. ingredients with benefits beyond meeting basic nutritional requirements, can play a crucial role thanks to their prophylactic effects. This study evaluated the effects of the supplementation of arginine, threonine and glutamine together with grape polyphenols on the gut integrity and functionality of broilers facing a stress condition. 108 straight-run newly hatched Ross PM3 chicks were kept until 35 days and were allocated to 3 treatments. Broilers in the control group were raised in standard conditions. In experimental groups, birds were administered with corticosterone in drinking water (CORT groups) to impair the global health of the animal and were fed a well-balanced diet supplemented or not with a mix of functional amino acids together with grape extracts (1 g/kg of diet—CORT + MIX group). Gut permeability was significantly increased by corticosterone in non-supplemented birds. This corticosterone-induced stress effect was alleviated in the CORT + MIX group. MIX supplementation attenuated the reduction of crypt depth induced by corticosterone. Mucin 2 and TNF-α gene expression was up-regulated in the CORT + MIX group compared to the CORT group. Caecal microbiota remained similar between the groups. These findings indicate that a balanced diet supplemented with functional AA and polyphenols can help to restore broiler intestinal barrier after a stress exposure.