Two studies were conducted to investigate the effect of
Bacillus amyloliquefaciens
CECT 5940 (BA) as a probiotic on growth performance, amino acid digestibility and bacteria population in broiler chickens under a subclinical necrotic enteritis (NE) challenge and/or fed diets with different levels of crude protein (CP). Both studies consisted of a 2 × 2 factorial arrangement of treatments with 480 Ross 308 mix-sexed broiler chickens. In study 1, treatments included 1) NE challenge (+/−), and 2) BA (1.0 × 10
6
CFU/g of feed) supplementation (+/−). In study 2, all birds were under NE challenge, and treatments were 1) CP level (Standard/Reduced [2% less than standard]) and 2) BA (1.0 × 10
6
CFU/g of feed) supplementation (+/−). After inducing NE infection, blood samples were taken on d 16 for uric acid evaluation, and cecal samples were collected for bacterial enumeration. In both studies, ileal digesta was collected on d 35 for nutrient digestibility evaluation. In study 1, the NE challenge reduced body weight gain (BWG), supressed feed conversion ratio (FCR) and serum uric acid levels (
P
< 0.001). Supplementation of BA increased BWG (
P
< 0.001) and reduced FCR (
P
= 0.043) across dietary treatments, regardless of challenge.
Bacillus
(
P
= 0.030) and
Ruminococcus
(
P
= 0.029) genomic DNA copy numbers and concentration of butyrate (
P
= 0.017) were higher in birds fed the diets supplemented with BA. In study 2, reduced protein (RCP) diets decreased BWG (
P
= 0.010) and uric acid levels in serum (
P
< 0.001). Supplementation of BA improved BWG (
P
= 0.001) and FCR (
P
= 0.005) and increased
Ruminococcus
numbers (
P
= 0.018) and butyrate concentration (
P
= 0.033) in the ceca, regardless of dietary CP level. Further, addition of BA reduced
Clostridium perfringens
numbers only in birds fed with RCP diets (
P
= 0.039). At d 35, BA supplemented diets showed higher apparent ileal digestibility of cystine (
P
= 0.013), valine (
P
= 0.020), and lysine (
P
= 0.014). In conclusion, this study suggests positive effects of BA supplementation in broiler diets via modulating gut microflora and improving nutrient uptake.