•Heterologous expression of two nitrite reductase subunits from Acidovorax wautersii.•Enzymatic activity range of temperature (20−60 °C) and pH (5.0–9.0).•Molecular dynamics simulations indicate specific amino residues of subunits.•Nitrite ion interacts with active site of enzyme by forming ligand hydrogen bonds. Large accumulation of nitrate in soil has resulted in soil salinization. Nitrate and nitrite reduction are of great importance for nitrogen assimilation, and understanding the specific roles of each participating reductase is necessary to describe the biochemical balance that dictates the bacteria responses to their environments. The genes encoding assimilatory nitrite reductase from Acidovorax wautersii QZ-4 were cloned and over-expressed in Escherichia coli. The molecular mass of over-expressed nitrite reductase was 91.75 kDa and 12.40 kDa for large subunit NirB and small subunit NirD, respectively. The purified recombinant enzyme showed broad activity range of temperature (20−60 °C) and pH (5.0–9.0). The optimum electron donor was MV + Na2S2O4, and NADH can be used as physiological electron donor. It was found that two important amino residues in NirD, Arg 90 and Arg 24 are critical for interaction with NirB, meanwhile, two amino residues of NirB such as Arg 257 and Asp 333 are found to be critical to interact with NirD. The nitrite ion was responsible for the unique interaction with the active site of NirB with the ligand quintuple hydrogen bonds with Arg 44 and Arg 128. These findings constitute a solid basis for valuable application of nitrite reduction in polluted environment, and give a reasonable explanation for the modes of action between enzyme from A. wautersii QZ-4 and nitrite ion.