The intestinal bacterial diversity of Stichopus japonicus was investigated using 16S ribosomal RNA gene (rDNA) clone library and Polymerase Chain Reaction/Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The clone library yielded a total of 188 clones, and these were sequenced and classified into 106 operational taxonomic units (OTUs) with sequence similarity ranging from 88 to 100%. The coverage of the library was 77.4%, with approximately 88.7% of the sequences affiliated to Proteobacteria. Gammaproteobacteria and Vibrio sp. were the predominant groups in the intestine of S. japonicus. Some bacteria such as Legionella sp., Brachybacterium sp., Streptomyces sp., Propionigenium sp. and Psychrobacter sp were first identified in the intestine of sea cucumber