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Various bacteria that adhere to the gut are important for the health of fish. Regulating the microbial community in a desirable direction may be beneficial in aquaculture for preventing and controlling the diseases caused by pathogenic microbes. In this study, we investigated the changes in the microflora in the intestinal tracts of turbot (Scophthalmus maximus) larvae after introducing a probiotic (Bacillus amyloliquefaciens) after the first feed. B. amyloliquefaciens was added as part of a nutrient enrichment system in live feed (Branchionus plicatilis or Artemia sinica), so it passed into the intestinal tracts of the newly hatched turbot larvae. The turbot larvae were fed on live feed containing B. amyloliquefaciens in the experimental group, whereas live feed without the probiotic was provided to larvae in the control group. The total bacterial genomic DNA in the larval guts was extracted and sequenced with an Illumina HiSeq PE250 system. According to the sequencing results, the abundances of microbial species and the microflora diversity were lower in the intestines in the experimental group than the control. Throughout development, the microflora structure in the intestines was mainly constructed before the first feed and the composition of the dominant operational taxonomic units (OTUs) was stable, where the abundances of OTU8, OTU124, OTU150, OTU107, and OTU17 were always high. Compared with the control, the structures of the microflora in the intestines were similar on different days during the development and the growth of larvae in the experimental group. However, the similarity of the microflora structure between different treatments was low on the same day. Furthermore, the mean proportion of common OTUs was only 74.7% in different treatments on each day, which indicates that the introduction of B. amyloliquefaciens in the live feed changed the microflora structure in the intestine. During the early development stage (days 3–30), the average abundance of Pseudomonas was reduced by 0.8% whereas that of Lactococcus increased by 3.5% in the experimental group. Pseudomonas spp. are considered potentially pathogenic bacteria but there is no direct evidence for the pathogenicity of Lactococcus in turbot. Moreover, several Lactococcus species are regarded as probiotics in aquaculture. Therefore, the use of B. amyloliquefaciens could be beneficial for optimizing the microbial community structure in the intestines of turbot larvae, which may explain the probiotic effect of B. amyloliquefaciens. This study provides a theoretical basis for the biological regulation of the microflora structure in the intestinal tract during turbot breeding. [ABSTRACT FROM AUTHOR]