A novel aerobic strain of Acinetobacter oleivorans AHP123 was isolated from activated sludge, which could conduct heterotrophic nitrification and denitrification simultaneously. This strain has excellent NH4+-N removal ability, with 97.93% removal rate at 24-hour. To identify the metabolic pathways of this novel strain, genes of gam, glnA, gdhA, gltB, nirB, nasA, nar, nor, glnK and amt were detected by genome analysis. Through RT-qPCR, it was found that the expression of key genes confirmed two possible ways of nitrogen removal in strain AHP123: nitrogen assimilation and heterotrophic nitrification aerobic denitrification (HNAD). However, the absence of some common HNAD genes (amo, nap and nos) suggested that strain AHP123 might have a different HNAD pathway from other HNAD bacteria. Nitrogen balance analysis revealed that strain AHP123 assimilated most of the external nitrogen sources into intracellular nitrogen.