Studies have shown that the addition of biochar to agricultural soils has the potential to mitigate climate change by decreasing nitrous oxide (N2O) emissions resulting from denitrification. Rice paddy field soils have been known to have strong denitrifying activity, but the response of microbes to biochar for weakening denitrification in rice paddy field soils is not well known. In this work, compared with the chemical fertilizer alone, the chemical fertilizer + 20 t hm(-2) biochar fertilizer slightly decreased denitrifying the nitrite reductase activity (S-NiR) and N2O emission without statistic difference, whereas the chemical fertilizer + 40 t hm(-2) biochar significantly boosted them. The abundance of nir-denitrifiers contributed to S-NiR and N2O emission, especially nirS-denitrifiers, rather than the variation of community structure. Pearson correlation analysis showed that NO2--N was a key factor for controlling the abundance of nir-denitrifiers, S-NiR and N2O emission. The biochar addition fertilization treatments strongly shaped the community structure of nirK-denitrifiers, while the community structure of nirS-denitrifiers remained relatively stable. In addition, Paracoccus and Sinorhizobium were revealed to be as the predominant lineage of nirS- and nirK-containing denitrifiers, respectively. Distance-based redundancy analysis (db-RDA) showed that changes in the nir-denitrifier community structure were significantly related to soil organic carbon, NO3--N, and total phosphorus. Our findings suggest that, although the nirS- and nirK-denitrifiers are both controlling nitrite reductase, their responses to biochar addition fertilization treatments showed significant discrepancies of diversity, abundance, and contribution to N2O and S-NiR in a paddy soil.