In view of the limited comparative research of pyrite-based iron autotrophic denitrification and sulfur autotrophic denitrification and low efficiency in nitrogen removal, pyrite was utilized as an electron donor for both sulfur autotrophic denitrification and iron autotrophic denitrification. Moreover, their performance in removing nitrogen from wastewater with a low carbon/nitrogen ratio (C/N) and the enhancement mechanisms involving heterotrophic bacteria were compared and investigated. The results show that the pyrite-based iron autotrophic denitrification system achieves higher nitrogen removal efficiency than the sulfur autotrophic denitrification system, and adding an appropriate amount of heterotrophic bacteria can enhance the nitrogen removal effect. At a heterotrophic-to-autotrophic sludge volume ratio of 1∶1, the pyrite-based iron autotrophic denitrification system yields effluent TN and NO^-3-N concentrations of 0.26 and 0 mg/L after 48 h, which were significantly lower than those of the sulfur autotrophic denitrification system (3.94 and 2.61 mg/L) under identical influent conditions. Thermomonas, Geothrix, Ferritrophicum, Acidovorax, Pseudomonas, Brachymonas, and other denitrifying bacteria can use reduced iron as an electron donor to participate in denitrification, and their relative abundance in the pyrite-based iron autotrophic denitrification system is all higher than that in the sulfur autotrophic denitrification system. Furthermore, the addition of heterotrophic sludge into the system can introduce Thauera . Compared with sulfur autotrophic bacteria, iron autotrophic bacteria and heterotrophic bacteria can exert a better synergistic denitrification and nitrogen removal effect.