The investigation of metabolic pathways and regulatory mechanisms in newly discovered species can offer valuable insights into the nitrogen removal function of heterotrophic nitrification–aerobic denitrification (HN-AD) bacteria. To investigate the nitrogen removal mechanism of a new genus, Delftia, we analyzed the complete genome, metabolic pathways, and the related genes of Delftia sp. B7. We further examined the nitrogen removal capacity of Delftia sp. B7 under various nitrogen sources and real wastewater. Our results demonstrate the presence of several genes in Delftia sp. B7, including narGHI, nasAB, nirK, nirS, nirBD, norBC, nosZ, nxrAB, gdhA, glnA, gltBD, amt, and nrt. These genes encode enzymes that facilitate ammonia assimilation, assimilatory nitrate reduction to nitrite, HN-AD, and dissimilatory nitrate reduction (DNRA) in Delftia sp. B7. Specifically, we propose an HN-AD pathway in Delftia sp. B7, NH4+-N → NH2OH → NO2−-N → NO3−-N → NO2−-N → NO → N2O → N2, which accounts for the majority of nitrogen removal. Here, the transformation of NH4+-N to NO2−-N was achieved by unknown enzymes or by another pathway. When treating municipal wastewater, Delftia sp. B7 was able to remove 45.62 ± 1.29% of TN. These findings provide a theoretical basis for utilizing microbial resources to mitigate nitrogen contamination.