The growth and nitrogen metabolism of heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria are affected by nitrite, but the mechanisms underlying this for strain Acinetobacter johnsonii EN-J1 are unclear. In this study, the addition of 10 mg/L nitrite increased the reduction rate of ammonium by 1.0 mg/L/h, and 20 mg/L nitrite increased the reduction rate of nitrate by 3.9 mg/L/h. Compared with the control, the nitrate reductase activity, electron transfer activity, and adenosine triphosphate content of EN-J1 were enhanced by 142.0%, 278.0% and 279.0%, respectively, in the nitrate removal process after the addition of 20 mg/L nitrite. The whole genome was annotated with nitrogen removal genes such as narGHI, narK, nsrR, nirBD, nasA, glnA, gltB, gdhA and amt. Transcriptome analysis showed that nitrite triggered significant upregulation of several key pathways, including nitrogen metabolism, the tricarboxylic acid cycle, and amino acid metabolism for enhancing denitrification. The expression of key denitrification genes (narG, narK, hmp, nirBD, glnA and nasA) was detected by real-time quantitative polymerase chain reaction. These results suggested that nitrite enhances denitrification by increasing the expression of denitrification genes, electron transfer and adenosine triphosphate levels, which is important for elucidating the mechanism of nitrite promotion of biological nitrogen removal efficiency.