In order to investigate the intestinal bacterial community comparison and its relationship with environmental factors, intestine samples were collected from pond and indoor tank-cultured Japanese flounder, Paralichthys olivaceus, and the composition, diversity and origin of bacterial community in them were investigated based on high throughput sequencing of 16S rRNA gene and six other samples from culture-associated environmental factors including culture water, feeds and pond sediment was also collected and their microbiota composition was also investigated using sequencing of 16S rRNA gene.The results showed that the dominant bacterial community harboring in pond-culture Japanese flounder intestine were Firmicutes(30.49%), Proteobacteria(19.16%), Fusobacteria(11.11%), wherein the most abundant bacteria was Bacillus(27.66%)and the least abundant was Vibrio based on genus level;whereas for pond-culture Japanese flounder, its intestine holds a core microbiota composed of Proteobacteria(44.31%), Firmicutes(11.57%), Actinobacteria(4.79%), in which the most abundant bacteria were Acinetobacter and Vibrio based on genus level.The most abundant bacterial operational taxonomic units(OTUs)in the Japanese flounder intestinal content are those related to nutrition metabolism, and the potential pathogens and probiotics are also important members of the intestinal microbiota.Differences and phylogenetic analysis revealed that the composition, diversity and origin of Japanese flounder intestinal bacterial communities were closely related with feed, followed by culture water.The present results would not only facilitate production of high-efficient probiotics, but also could provide theoretical support for construction of practical micro-ecological technology for P.olivaceus under different culture environment.