An expanded-granular sludge bed (EGSB) reactor was set-up with artificial water by seeding a 60–d stored ANAMMOX sludge. The nitrogen removal efficiency of ANAMMOX enrichment culture in the reactor was determined. In addition, the main microbial populations and the relative abundance of ANAMMOX bacteria were investigated by molecular approaches. Results show that the maximum nitrogen removal rate was 3.0 kg-N·m-3·d-1 after 185 d, and the ammonium and nitrite removal efficiencies were all over 85%. Analysis of 16S rRNA gene-cloning indicates that the main microbial population in the ANAMMOX enrichment culture was changed from Candidatus Brocadia fulgid and Candidatus Brocadia brasiliensis (0 day) to Candidatus Jettenia asiatica (185 day). Fluorescence in situ hybridization analysis shows that the relative abundance of ANAMMOX bacteria was increased from (57.69±4.79)% to (83.32±4.40)%. The results of qPCR further indicate that the gene copies of ANAMMOX bacteria in the granules were increased from 1.14×1011copies/g wet weight to 3.69×1011copies/g wet weight.