The study investigated the denitrification effect of the iron autotrophic denitrification process for removing nitrite under anaerobic conditions, utilizing sponge iron as the electron donor. When the C/N ratio equaled 1, defined as the ratio of chemical oxygen demand to total nitrogen (TN), and the influent nitrite nitrogen (NO2--N) was at 80 mg/L, the average steady-state TN effluent concentration of this system was 41.94 mg/L during the 79-day experiment. The TN value exhibited a significant decrease compared to both the sponge iron system (68.69 mg/L) and the carbon source system (56.50 mg/L). Sponge iron is beneficial for providing an electron donor and ensuring an anaerobic system, fostering an environment that promotes microorganism growth while effectively inhibiting the conversion of nitrite to nitrate. In addition, carbon sources play a vital role in ensuring microorganism growth and reproduction, thereby aiding in TN removal. The optimal parameters based on the effectiveness of TN removal in the iron autotrophic denitrification system were determined to be s-Fe0 dosage of 30 g/L and C/N = 1.5. These results suggest that the iron autotrophic denitrification process, driven by sponge iron, can effectively remove nitrite under anaerobic conditions.