Ammonia-oxidizing bacteria (AOB) play a key role in nitrogen-removal wastewater treatment plants (WWTPs) as they can transform ammonia into nitrite. AOB can be enriched in activated sludge through autotrophic domestication although they are difficult to be isolated. In this study, autotrophic domestication was carried out in a lab-scale sequencing-batch-reactor (SBR) system with two activated sludge samples. The ammonia removal capacity of the sludge samples increased during the domestication, and pH exhibited a negative correlation with the ammonia removal amount, which indicated that it was one important factor of microbial ammonia oxidation. The count of AOB, measured by the most probable number (MPN) method, increased significantly during autotrophic domestication as ammonia oxidation efficiency was enhanced. We investigated the changes in the community structure of AOB before and after domestication by amoA clone library and T-RFLP profile. It showed that AOB had been successfully enriched and the community structure significantly shifted during the domestication. Two groups of AOB were found in sludge samples: Nitrosomonas-like group remained predominant all the time and Nitrosospira-like group changed obviously. Simultaneously, the total heterotrophic bacteria were investigated by MPN and Biolog assay. The metabolic diversity of heterotrophs had changed minutely, although the count of them decreased significantly and lost superiority of microbial communities in the sludge.