Nitrite (NO2 (-)-N) accumulation in denitrification can provide the substrate for anammox, an efficient and cost-saving process for nitrogen removal from wastewater. This batch-mode study aimed at achieving high NO2 (-)-N accumulation over long-term operation with the acetate as sole organic carbon source and elucidating the mechanisms of NO2 (-)-N accumulation. The results showed that the specific nitrate (NO3 (-)-N) reduction rate (59.61 mg N VSS-1 h(-1) at NO3 (-)-N of 20 mg/L) was much higher than specific NO2 (-)-N reduction rate (7.30 mg N VSS-1 h(-1) at NO3 (-)-N of 20 mg/L), and the NO2 (-)-N accumulation proceeded well at the NO3 (-)-N to NO2 (-)-N transformation ratio (NTR) as high as 90 %. NO2 (-)-N accumulation was barely affected by the ratio of chemical oxygen demand (COD) to NO3 (-)-N concentration (C/N). With the addition of NO3 (-)-N, NO2 (-)-N accumulation occurred and the specific NO2 (-)-N reduction rate declined to a much lower level compared with the value in the absence of NO3 (-)-N. This indicated that the denitrifying bacteria in the system preferred to use NO3 (-)-N as electron acceptor rather than use NO2 (-)-N. In addition, the Illumina high-throughput sequencing analysis revealed that the genus of Thauera bacteria was dominant in the denitrifying community with high NO2 (-)-N accumulation and account for 67.25 % of total microorganism. This bacterium might be functional for high NO2 (-)-N accumulation in the presence of NO3 (-)-N.