Abstract: The gut microbiota is essential in shaping many of its host’s functional attributes. The gut microbiota is necessary for the proper physiological development of the gut and for the animal’s ability to digest and convert plant mass into food products. Herein, based on high throughput sequencing technology MiSeq, the structure of intestinal bacterial colonies of Apostichopus japonicus cultured in Dalian, Yantai and Fujian was analyzed. The results showed that the units of classification operation (OTUs) of the four samples were 455, 542, 440 and 242, respectively. Both the species abundance and dominant strains were quite different among the four samples. The maximum of flora’s species abundance was 4.78 in Yantai, followed by 4.47 in Dalian, 4.46 in Putian and 2.47 in Xiapu. And, the flora’s species abundance of Putian pond was more than twice as high as that of the Xiapu sea cage. A total of 317 bacterial species were detected in the four producing areas. The hindgut dominant bacteria was Lutibacter (10.13%, Dalian), Haliea, Halioglobus (21.34%, 12.58%, Yantai), Lactococcus (24.5%, Putian) and Formosa (25.09%, Xiapu). In a word, this study aimed at providing basic information for further research on the relationship between A. japonicu intestinal flora and its feeding, digestion and immunity.