We investigated the changes in communities of bacteria, ammonia-oxidizing bacteria, and Nitrospira during the operation of a pufferfish Takifugu 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 pyrosequencing. The relative abundance of Proteobacteria (Gammaproteobacteria) increased, while that of Bacteroidetes (Flavobacteria) decreased. The proportions of Nitrosomonas and Nitrospina 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 units were related to Nitrosomonas 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 strain Nitrospira sp. Ecomares 2.1 increased, but that of the abundant species Nitrospira marina decreased. Our results demonstrated that the communities of bacteria, ammonia-oxidizing bacteria, and Nitrospira were changing during the operation of the pufferfish recirculating aquaculture system.