Abstract The effects of FeS on nitrogen removal performance and microbial community of anammox process were studied. During the start-up period, the removal efficiencies of nitrite and total nitrogen were significantly improved by FeS. The addition of FeS increased the content of iron ions in the reactor and promoted the synthesis of heme c, which was involved in the formation of various enzymes. Compared with the control, the abundance of anammox bacteria in the FeS reactor was increased by 29%, and the expression level of the nirS gene (encoding cd1 type nitrite reductase containing heme) was nearly doubled. The content of nitrite reductase (ammonia-forming) in the community was increased by 26.4%. The difference in functional bacteria and enzyme contents in the microbial community resulted in a difference in nitrogen removal rate (NRR) between the two reactors. High-throughput results indicated that FeS increased the richness and diversity of microbial community and enhanced the metabolic function of the microbial community. The addition of FeS did not change the dominant position of Ca. Kuenenia in both reactors. But the relative abundance of heterotrophic denitrifying bacteria was reduced with FeS, which may be related to the inhibition effect of S2− produced by FeS.