The soybean endosymbiont Bradyrhizobium japonicum is able to scavenge the greenhouse gas N2O through the N2O reductase (Nos). In previous research, N2O emission from soybean rhizosphere was mitigated by B.japonicumNos(++) strains (mutants with increased Nos activity). Here, we report the mechanism underlying the Nos(++) phenotype. Comparative analysis of Nos(++) mutant genomes showed that mutation of bll4572 resulted in Nos(++) phenotype. bll4572 encodes NasS, the nitrate (NO3-)-sensor of the two-component NasST regulatory system. Transcriptional analyses of nosZ (encoding Nos) and other genes from the denitrification process in nasS and nasST mutants showed that, in the absence of NO3-, nasS mutation induces nosZ and nap (periplasmic nitrate reductase) via nasT. NO3- addition dissociated the NasS-NasT complex in vitro, suggesting the release of the activator NasT. Disruption of nasT led to a marked decrease in nosZ and nap transcription in cells incubated in the presence of NO3-. Thus, although NasST is known to regulate the NO3--mediated response of NO3- assimilation genes in bacteria, our results show that NasST regulates the NO3--mediated response of nosZ and napE genes, from the dissimilatory denitrification pathway, in B.japonicum.