The nitrate-dependent anaerobic ferrous oxidation (NAFO) is a newly discovered biological denitrification process. However, the formation of iron crusts on the surface of microorganisms by residual ferric iron poses a significant challenge to the maintenance of NAFO performance. To solve the aforementioned issue, this study set up two continuous NAFO systems with (R2) and without EDTA-2Na (R1), and investigated the impact of EDTA-2Na on nitrate removal and microbial community. After 91 days of operation, EDTA-2Na demonstrated the ability to improve the ferrous utilization rate in NAFO process, and the average effluent ferrous concentration was 15.71 mg/L in R2, which was superior to the 50.03 mg/L of R1. Compared with the conventional system, R2 increased nitrate removal by 17 % and stabilized the growth of microorganisms by mitigating iron crusts in the long-term operation. Batch experiments indicated that the optimal nitrate removal efficiency was achieved at an initial Fe/N of 3.24 and a pH of 6.42. High-throughput analysis implied that the reactor with EDTA-2Na contained numerous nitrogen removal pathways. This study provided a practical method for the long-term stable operation of a NAFO system.