To evaluate the effect of Fe3+ on nitrogen (N) removal and associated microbial characteristics during simultaneous chemical phosphorus (P) removal, a sequencing batch reactor was used to analyze the changes in the microbial community and metabolic pathways caused by Fe3+ addition. Results demonstrated that Fe3+ promoted ammonia nitrogen (NH4+-N) removal and inhibited denitrification process, and increased the sludge particles (D50) and the biomass per sludge particle size. Furthermore, the abundances of denitrifying bacteria (Haliangium and Terrimonas) and biological phosphorus removing bacteria (Halaingium, norank_f_Saprospiraceae and SM1A02) were decreased. On the contrary, the increase of nitrifying bacteria abundance and the coding genes of nitrification-related enzymes confirmed the promotion for nitrification with Fe3+ addition. Besides, Fe3+ inhibited the interspecific relationship between denitrifying bacteria genera and other genera to reduce denitrification efficiency.