To investigate the effect and mechanism of Fe-humic acid complex (Fe-HA) on anaerobic ammonia oxidation (ANAMMOX) reactors, the differences in nitrogen removal performance, sludge characteristics and microbial community structure between RFe-HA with Fe-HA complex and RCON without Fe-HA complex were compared. The results showed that after the nitrogen load was increased (21 d), the NH4+-N and NO2--N removal efficiencies of RFe-HA were 93.28% and 88.60%, respectively, which were higher than those of RCON. After the operation was stabilized, the nitrogen removal performance of RFe-HA and RCON was basically the same. The sludge characteristics showed that the specific anaerobic ammonia oxidation activity (SAA) of granular sludge within RFe-HA was less than RCON, while the sludge concentration was higher than RCON. The average particle size and the percentage of granular sludge >0.85 mm were also higher in RFe-HA than in RCON. Compared to RCON, the granular sludge in RFe-HA had higher extracellular polymeric substances (EPS) fluorescence intensity and higher content of hydrophobic tryptophan-like substances in the EPS fraction. The community structure showed that the abundance of Candidatus Kuenenia in RFe-HA was 26.35% at 30d, which was higher than that of 23.58% in RCON. The addition of Fe-HA complex promoted the proliferation of Candidatus Kuenenia and caused an increase in the secretion of EPS and tryptophan-like substances from granular sludge. On the one hand, the increase of EPS and tryptophan-like substances promoted the formation of granular sludge, increased particle size, increased sludge concentration, and enhanced nitrogen removal performance of the reactor when the nitrogen load changes; on the other hand, they blocked the mass transfer efficiency between microorganisms and substrate and reduced the SAA of granular sludge. However, the increased sludge concentration compensated for the loss of nitrogen removal due to lower SAA, resulting in essentially the same nitrogen removal performance of RFe-HA and RCON at stable operation.