To investigate the changes of the gut microbiota in grass carp (Ctenopharyngodon Idella), grass carps were administrated with different dosages of Bacillus subtilis spores (B.s-CotC-CsPmy)(1×105, 1×108, 1×1011 CFU/g pellets). And the fishes were named BL, BM, BH groups, respectively. Grass carp administrated with B.s-CotC (BC group) and without spores(NC group) were considered as control. 8 weeks after the beginning of the administration, three fishes in each group were euthanized as mentioned above and sampled. The whole intestines were aseptically excised and opened. They were rinsed with sterilized PBS to remove the contents. Intestines from three fishes in the same group were collected into one sterile centrifuge tube. The intestinal samples were homogenized with sterilized PBS. The genomic DNA was extracted using the OMEGA D3350 Bacterial DNA Kit (Omega, USA) according to manufacturer’s protocols. The V3-V4 regions of 16S ribosomal DNA of bacteria were amplified, purified and pooled in equimolar and paired-end sequenced (2 × 250 bp) on an Illumina MiSeq platform