This study clarified the dominant nitrogen (N)-transformation pathway and the key ammonia-oxidizing microbial species at three loading levels during optimization of the anoxic/oxic (A/O) process for sewage treatment. Comprehensive N-transformation activity analysis showed that ammonia oxidization was performed predominantly by aerobic chemolithotrophic and heterotrophic ammonia oxidization, whereas N-2 production was performed primarily by anoxic denitrification in the anoxic unit. The abundances of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria, and anaerobic AOB in activated sludge reflected their activities on the basis of high-throughput sequencing data. AOB amoA gene clone libraries revealed that the predominant AOB species in sludge samples shifted from Nitrosomonas europaea (61% at the normal loading level) to Nitrosomonas oligotropha (58% and 81% at the two higher loading levels). Following isolation and sequencing, the predominant culturable heterotrophic AOB in sludge shifted from Agrobacterium tumefaciens (42% at the normal loading level) to Acinetobacter johnsonii (52% at the highest loading level).