| Summary: | The denitrification process is one of the important processes in the soil nitrogen (N) cycle, and is closely related to the loss of soil N fertilizer. Five treatments were included in this study: N0 (control, no N application); N90 (N application rate 90 kg ha<sup>−1</sup>); N150 (N application rate 150 kg ha<sup>−1</sup>); N210 (N application rate 210 kg ha<sup>−1</sup>); and N270 (N application rate 270 kg ha<sup>−1</sup>), to study the effect of different N application rates on the soil <i>nirS</i>-type denitrifying bacterial community structure, the influence of key enzyme activities during the denitrification process, and the main environmental factors affecting the variation of the denitrifying bacterial community in maize field soil under the mulched fertigation system in the semi-arid region of Northeast China. The results showed that increasing N fertilizer application significantly increased the contents of soil inorganic N and acid-hydrolyzable organic N, but significantly decreased pH. N fertilizer significantly increased nitrate reductase (NAR) activity and nitrite reductase (NIR) activity. Excessive application of N fertilizer significantly increased the <i>nirS</i> gene copy numbers, and, at the same time, significantly decreased the diversity of <i>nirS</i>-type soil denitrifying bacteria. Proteobacteria was the dominant denitrifying phylum in all treatments, and N application promoted the growth of <i>Bradyrhizobium</i> belonging to this phylum. The application of N fertilizer significantly changed the community structure of <i>nirS</i> denitrifying bacteria, and the NO<sub>3</sub><sup>−</sup>-N content was the most important factor for this observation. Soil organic matter (SOM) and non-hydrolyzable N (NHN) indirectly affected the denitrifying bacterial community structure through NAR activity and NIR activity, while soil total N (TN) and nitrate N (NO<sub>3</sub><sup>−</sup>-N) indirectly affected yield through denitrifying bacterial community structure. Although increasing N fertilizer was beneficial in increasing soil nutrients, the community structure of <i>nirS</i>-type denitrifying bacteria changed significantly. This is attributed to the increase in soil NO<sub>3</sub><sup>−</sup>-N accumulation caused by a large amount of N application. The results of this research provide an important scientific basis for further research on the response mechanism of farmland soil denitrifying microorganisms to different N fertilizer managements under the mulched fertigation system in the semi-arid region of Northeast China.
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