We investigated the changes in communities of bacteria, ammonia-oxidizing bacteria, andNitrospira during the opera-tion of a pufferfishTakifugu rubripes recirculating aquaculture system by using high-throughput DNA sequencing. Differences in bacterial communities were observed at days 1–32, 47–62 and 78–93 of biofilm development by using 16S rRNA gene pyrose-quencing. The relative abundance of Proteobacteria (Gammaproteobacteria) increased, while that of Bacteroidetes (Flavobacteria) decreased. The proportions ofNitrosomonasandNitrospina ranged from 0.02％ to 0.30％ and from 0.02％ to 0.83％, respectively. Ammonia monooxygenase gene pyrosequencing revealed that the top three operational taxonomic unitswere related toNitrosomonas aestuarii (17.5％–61.1％), uncultured beta proteobacterium clone B67S-54 (1.9％–45.2％), and uncultured bacterium clone AZPa8 (3.6％–24.7％). Nitrite oxidoreductase gene pyrosequencing revealed that the relative abundance of the dominant strainNitrospira sp. Ecomares 2.1 increased, but that of the abundant speciesNitrospira marina decreased. Our results demonstrated that the communities of bacteria, ammonia-oxidizing bacteria, andNitrospira were changing during the operation of the pufferfish recirculating aquacul-ture system.