Exploring the effects of nitrate ions (NO3-) on the bio-oxidation of Fe2+ and pyrite will help reveal the actual mechanism of acid mine drainage (AMD) production. Long period shaking flask experiments were carried out in order to assess the effect of NO3- on the Acidithiobacillus ferrooxidans LX5 (A. ferrooxidans LX5)-mediated bio-oxidation of Fe2+ and pyrite. In Fe2+ bio-oxidation systems, A. ferrooxidans LX5 had stronger Fe2+ oxidation capabilities in a NO3--loaded solution than in a NO3--free solution after 24 days, and the Fe2+ bio-oxidation capacity of A. ferrooxidans LX5 acclimatized in solutions containing low concentrations (8.2-32.9 mmol/L) of NO3- was greater than when it was acclimatized in high NO3- concentration solutions (49.4-65.8 mmol/L). In pyrite bio-oxidation systems, in comparison with the system without NO3-, pyrite bio-oxidation efficiency was significantly increased when the NO3- concentration in the system was 8.2-16.5 mmol/L, and that the pyrite bio-oxidation efficiency in the system containing 8.2 mmol/L of NO3- was greater than that for the system with 16.5 mmol/L of NO3-. The pyrite bio-oxidation efficiency was inhibited when the NO3- concentration was above 32.9 mmol/L. The results from this study can be used to reveal the actual control behavior of NO3- on AMD production.