Fe(II) is one of the commonly used additives in wastewater treatment and proved to be beneficial for promoting microbial activity. In this study, the effects of Fe(II) on the specific anammox activity (SAA) and reactor performance were proved to be concentration-dependent. In the short-term experiment, low concentration of Fe(II) (5-80 mg/L) significantly enhanced the SAA, while high concentration of Fe(II) (120-300 mg/L) inhibited the SAA. It was confirmed that anammox can be domesticated after long-term exposure to low Fe(II) concentration, and the SAA could be further enhanced by higher Fe(II) concentration in the following phases. In addition, as an important factor for anammox granulation and maintaining the SAA, the extracellular polymeric substance (EPS) was also affected by Fe(II) addition. In spite of the effects on SAA and EPS, Fe(II) was proved to be the key factor that enhances the N 2 O emission via abiotic pathway in the anammox reactor. © 2021 Water Environment Federation • Practitioner points • Low Fe(II) concentrations enhanced SAA, while high concentrations inhibited SAA. • Long-term acclimatization by Fe(II) improved the tolerance of anammox to Fe(II). • Fe(II) affects the amount and constituent of EPS and the performance of anammox granules. • Accumulation of Fe(II) in the AAFEB reactor promoted the N 2 O emission.