In this study, the effect of Fe (II) on Anaerobic Ammonium Oxidation (ANAMMOX) process was investigated by step-wise increasing the Fe (II) in influent from 1 to 50 mg L−1. The nitrogen removal, biofilm property and the microbial community were analyzed in each phase. Results showed that, the anaerobic ammonia-oxidizing bacteria (AAOB) bioactivity and the nitrogen removal of ANAMMOX system were slightly improved to 0.58 from the initial 0.51 kg m−3 d−1 by Fe (II) in 1–5 mg L−1. The nitrogen removal was suppressed and could recover to the initial level during the same period under 10–20 mg L−1 Fe (II), while it did not recover to the initial level under 30 mg L−1 Fe (II) and showed no recovery performance under 50 mg L−1 Fe (II). The irreversible suppression threshold of Fe (II) was calculated as 50 mg L−1. The iron content in ANAMMOX biofilm presented linear correlation with the influent Fe (II) in 1–20 mg L−1, which then tended to be stable when Fe (II) was higher. Dehydrogenase activity (DHA) showed similar and faster response to Fe (II) than the microbial activity, and it was an effective pre-indicator for the nitrogen removal performance in the ANAMMOX system suffered Fe (II). The Fe (II) feeding firstly led to the relative abundance of AAOB decreased to 11.04% from the initial 35.46%, and finally picked up to 19.39% after the long-term acclimatization. •Fe (II) in 1–5 mg L−1 effectively improved AAOB bioactivity and nitrogen removal.•Fe (II) in 10–30 mg L−1 performed reversible inhibition on ANAMMOX process.•The irreversible suppression threshold of Fe (II) on ANAMMOX was 50 mg L−1.•DHA was a pre-indicator for nitrogen removal performance of ANAMMOX system suffered Fe (II).•Fe (II) feeding lowered the relative abundance of Candidatus Kuenenia.