Background:The ban of in-feed antimicrobial additives has negatively affected the poultry industry by causing necrotic enteritis (NE) to emerge in the flocks. Alternatives such as Bacillus probiotics have shown to be effective on eliminating the negative effects of this disease. Two experiments were conducted to investigate the effect of Bacillus amyloliquefaciens CECT 5940 (BA) in broiler chickens under NE challenge and/or fed diets with different protein levels. Methods:In both experiments, 480 day-old mix-sexed Ross-308 broilers were arranged in a 2 × 2 factorial arrangement of treatments. In experiment 1, the factors were NE challenge (yes or no) and probiotic (yes or no). In experiment 2, the factors were dietary crude protein levels (standard or reduced) and probiotic (yes or no) and were used under NE challenge condition. Oral administration of Eimeria oocysts (day 9) followed by inoculation with Clostridium perfringens (day 14 and 15) was used to induce NE challenge. On day 16, two birds from each treatment were gavaged with fluorescein isothiocyanate-dextran (FITC-d) and blood samples were collected for gut integrity evaluation, and jejunal samples were collected for gene expression assay. (Continued on next page) (Continued from previous page) Results:In experiment 1, BA supplementation decreased caspase-3 (CASP3) (P<0.001) and caspase-8 (CASP8) (P<0.05) and increased occludin (OCLD) (P<0.05) expression regardless of the challenge. Additionally, BA supplementation downregulated interfron-γ(IFN-γ) expression (P<0.01) and upregulated immunoglobulin-G (IgG) (P<0.01) and immunoglobulin-M (IgM) (P<0.05) only in challenged birds. In experiment 2, the expression of genes encoding mucin-2 (MUC2) (P<0.001), tight junction protein-1 (TJP1) (P<0.05) and OCLD (P<0.05) were upregulated by the addition of BA in the diet, regardless of the crude protein level. Further, BA supplementation downregulated INF-γ(P<0.01) and upregulated immunoglobulin-A (IgA) (P<0.05), IgM (P<0.05) and IgG (P<0.01) regardless of the crude protein level. Conclusion:These findings suggest that supplementation of BA in broiler diets can improve gut health by modulation of genes related to the mucosal barrier, tight junction, and immunity in broilers challenged by unfavourable conditions such as NE challenge.