Zero-valent iron (ZVI) was used to improve the biological autotrophic denitrification process between nitrate and ammonia by anaerobic ammonia oxidation ( ANAMMOX) bacteria. With the addition of ZVI, the biological autotrophic denitrification process could be reacted in the influent condition of pH was 7-8, at 35°C ±0.5°C, the concentration of ammonia was 50-100 mg · L⁻¹ and the concentration of nitrate was 50-100 mg · L⁻¹. The highest conversion rate could be reached to 17.2 mg · (L·h) ⁻¹. With the change of reaction time and the molar ratio of nitrate and ammonia in influent, the final molar conversion ratio of nitrate and ammonia in effluent fluctuated between 1.2-3. 5. The result showed that this autotrophic denitrification process was not belonged to elementary reaction. The mechanism of this autotrophic denitrification process could be summarized that with the reduction of ZVI, the nitrate could be reduced to nitrite. Hereafter, the ANAMMOX process reacted between the nitrite and ammonia.