This study provided a deep insight into the impacts of trace elements (Mn2+, Zn2+ and Cu2+) on nitritation-anammox process. For short-term exposure, all the three elements could improve the nitrogen removal rate (NRR) and the optimal concentrations were 2.0 mg/L, 2.0 mg/L and 0.5 mg/L for Mn2+, Zn2+ and Cu2+, respectively. Accordingly, the NRR were enhanced 54.62%, 45.93% and 44.09%. The long-term experiments were carried out in lab-scale sequencing batch reactors. The surprising results showed that only Mn2+ addition could enhance the long-term nitritation-anammox process, and the NRR increased from 0.35 ± 0.01 kg N/m3/d (control, no extra trace element addition) to 0.49 ± 0.03 kg N/m3/d. Vice versa, the amendment of Zn2+ reduced the NRR to 0.28 ± 0.02 kg N/m3/d, and Cu2+ had no significant effect on the NRR (0.36 ± 0.01 kg N/m3/d). From the analysis of microbial community structure, it was explained by the increasing abundance of anaerobic ammonium oxidizing bacteria (AnAOB) only in Mn2+ treatment, whereas Zn2+ predominantly promoted ammonium oxidizing bacteria (AOB). Additionally, the majority of Mn2+ was identified inside AnAOB cells, and Zn2+ and Cu2+ were mainly located in AOB. Our results indicated the synergistic effects of trace elements on nitritation-anammox, both short-term encouraging activities of AnAOB and long-term altering microbial community structure. This work implies the importance of trace elements addition in nitritation-anammox process.