Iron humic acid (Fe-HA) has been proved that it can enhance the process of microbial metabolism for a wide range of pollutants. The effect of Fe-HA on the anaerobic ammonia oxidation (ANAMMOX) process during the increase of nitrogen loading rate (NLR) were investigated, and the potential mechanism of Fe-HA on the ANAMMOX process was explored through the analysis of nitrogen removal performance, sludge characteristics, and microflora structure. The results show that the nitrogen removal performance of R_Fe-HA is higher than that of R_CON during the initial stage after the NLR is increased. During the stabilization stage after the NLR is increased, the nitrogen removal performance of R_Fe-HA and R_CON is almost the same. The specific ANAMMOX activity (SAA) of R_Fe-HA is less than R_CON, while the sludge concentration, average particle size, and percentage of particle size >0.85 mm are higher than R_CON. Furthermore, R_Fe-HA has a higher concentration of extracellular polymeric substances (EPS) and a higher content of hydrophobic tryptophan-like substances in the EPS fraction. The abundance of Candidatus Kuenenia is higher in R_Fe-HA than R_CON at 30 d. The addition of Fe-HA promoted the proliferation of Candidatus Kuenenia and cause an increase in the secretion of EPS and tryptophan-like substances from granular sludge. The increase of EPS and tryptophan-like substances promoted the formation of granular sludge, increased sludge concentration and particle size, and enhanced nitrogen removal performance of the reactor during the initial stage of the NLR increase. However, the increase of EPS blocked the mass transfer efficiency between microorganisms and substrate and reduce the SAA of granular sludge.