The potential of a novel class of prebiotics, arabinoxylan-oligosaccharides (AXOS), was investigated on growth performance and gut microbiota of juvenile Acipenser baerii. Two independent feeding trials of 10 or 12 weeks were performed with basal diets supplemented with 2% or 4% AXOS-32-0.30 (trial 1) and 2% AXOS-32-0.30 or AXOS-3-0.25 (trial 2), respectively. Growth performance improved by feeding 2% AXOS-32-0.30 in both trials, although not significantly. Microbial community profiles were determined using 454-pyrosequencing with barcoded primers targeting the V3 region of the 16S rDNA. AXOS significantly affected the relative abundance of bacteria at the phylum, family, genus, and species level. The consumption of 2% AXOS-32-0.30 increased the relative abundance of Eubacteriaceae, Clostridiaceae, Streptococcaceae and Lactobacillaceae while the abundance of Bacillaceae was greater in response to 4% AXOS-32-0.30 and 2% AXOS-3-0.25. The abundance of Lactobacillus spp. and Lactococcus lactis was greater after 2% AXOS-32-0.30 intake. Redundancy analysis showed a distinct and significant clustering of the gut microbiota of individuals consuming an AXOS diet. In both trials, concentration of acetate, butyrate and total short chain fatty acids increased in fish fed 2% AXOS-32-0.30. Our data demonstrate a beneficial shift in the hindgut microbiome of fish consuming different preparation of AXOS, with potential application as prebiotics. This article is protected by copyright. All rights reserved.