Although diverse microorganisms can remove ammonium and nitrate simultaneously, their metabolic mechanisms are not well understood. Paracoccus denitrificans R-1 showed the maximal NH 4 + removal rate 9.94 mg L -1 ·h -1 and 2.91 mg L -1 ·h -1 under aerobic and anaerobic conditions, respectively. Analysis of the nitrogen balance calculation and isotope tracing experiment indicated that NH 4 + was consumed through assimilation. The maximal NO 3 - removal rate of strain R-1 was 18.05 and 19.76 mg L -1 ·h -1 under aerobic and anaerobic conditions, respectively. The stoichiometric consumption ratio of acetate to nitrate was 0.902 and NO 3 - was reduced to N 2 for strain R-1 through 15 NO 3 - isotopic tracing experiment, which indicated a respiratory process coupled with the oxidation of electron donors. Genomic analysis showed that strain R-1 contained genes for ammonium assimilation and denitrification, which effectively promoted each other. These findings provide insights into microbial nitrogen transformation and facilitate the simultaneous removal of NH 4 + and NO 3 - in a single reactor.