The lack of seed sludges for Ammonium Oxidizing Bacteria (AOB) and slow-growing ANaerobic AMMonium OXidation (ANAMMOX) bacteria is one of the major problem for large-scale application. In this study, 24m3 of single-stage SBR (Sequencing Batch Reactor) was operated to remove nitrogen from reject water using AOB and ANAMMOX bacteria cultivated from activated sludge in the field. The ANAMMOX activity was found after 44 days of cultivation in the ANAMMOX cultivation reactor, and then 0.66 kg N/m3/d of the nitrogen removal rate was achieved at 0.78 kg N/m3/d of the nitrogen loading rate at 153 days of cultivation. The AOB cultivation reactor showed 0.2 kg N/m3/d of nitrite production rate at 0.4 kg N/m3/d of nitrogen loading rate after 36 days of operation. The cultivated ANAMMOX bacteria and AOB was mixed into the single-stage SBR. The feed distribution was applied to remove total nitrogen stably in the single-stage SBR. The nitrogen removal rate in the single-stage SBR was gradually enhanced with an increase of specific activities of both AOB and ANAMMOX bacteria by showing 0.49 kg N/m3/d of the nitrogen removal rate at 0.56 kg N/m3/d of the nitrogen loading rate at 54 days of operation.