•Acinetobacter junii YB was isolated from activated sludge and identified.•The strain exhibited efficient removal of ammonium at varying culture conditions.•Hydroxylamine, nitrite and nitrate could be utilized by strain YB.•The strain showed capacities for aggregation and hydrophobicity. A novel heterotrophic nitrifying bacterium was isolated from activated sludge and was identified as Acinetobacter junii YB. The strain exhibited efficient heterotrophic nitrification–aerobic denitrification ability at a broad range of ammonium loads and had the capability to utilize hydroxylamine, nitrite and nitrate as a sole nitrogen source. Based on the nitrogen removal and enzyme assay, the nitrogen removal pathway was speculated to be achieved through heterotrophic nitrification coupled with aerobic denitrification. In addition, single-factor experiments showed that efficient heterotrophic nitrification and growth of strain YB occurred with succinate as the carbon source, pH 7.5, 37°C, and high C/N ratio and dissolved oxygen. Furthermore, the new isolate showed capacities for aggregation and hydrophobicity. Regular variations of the flocculating ability and relative hydrophobicity were observed during the whole cultivation. The ability to perform heterotrophic nitrification–aerobic denitrification and cell aggregation demonstrated the great potential of the strain YB for future applications.