Chinese mitten crab (Eriocheir sinensis) is one of the important economic species of freshwater aquaculture in China. Dietary astaxanthin affects the number and composition of culture-based dominant bacteria from intestinal tract and gills, which is closely linked to the body's immunity. In order to realize the relationship between dietary astaxanthin and the balance of bacteria in intestinal tract and gills, five diets with various concentration astaxanthin (0.00, 26.60, 41.62, 81.37 and 75.35 mg/kg, respectively) were fed to adult male crabs for 70 days. The dominant bacteria number and composition of five crab groups were measured by culture-based enumeration techniques, and then the isolates were sequenced by 16S rRNA gene analysis. A total of 106 bacterial strains were isolated from the intestinal tract, gills and aquaculture water. 92 bacterial strains were dominant bacteria, of which 29 bacterial strains were from intestinal tract, 32 bacterial strains from gills and 31 bacterial strains from aquaculture water. At the genus level, the dominant bacteria were Citrobacter, Pseudomonas and Aeromonas in the intestinal tract, by contrast, the dominant bacteria were Citrobacter, Bacillus, Pseudomonas and Aeromonas in the gill, and the dominant bacteria were Bacillus, Aeromonas, Citrobacter, Pseudomonas and Chryseobacterium in the aquaculture water. The total number of culture-based dominant bacteria in intestinal tract was the highest (1.06×10⁸ cfu/g) in group 2. The number of potential pathogenic bacteria of group 5 (75.35 mg/kg) was significantly decreased in intestinal tract. However, the total number of dominant bacteria of group 5 was significantly higher than the other four groups in the gill, and the numbers of dominant bacteria had no significant difference in all five groups in aquaculture water. To conclude, dietary astaxanthin with various concentrations could significantly affect the culture-based dominant bacteria composition of intestinal tract and gill in male E. sinensis. The number of potentially pathogenic bacteria in intestinal tract and gills accounted for high proportion in group 3. This study for the first time analyzed the effect of astaxanthin on number and composition of culture-based dominant bacteria of intestinal tract and gills in male Chinese mitten crab, and discussed the relationship between concentration of dietary astaxanthin, aquaculture water and the number of culture-based dominant bacteria from intestinal tract and gill, and the results may provides a theoretical basis for E. sinensis diet exploitation and bacterial flora control in the future.