[Objective] to investigate the influence of acidophilic heterotrophic bacteria on Acidithiobacillus ferrooxidans in extremely acidic environment such as acid mine drainage (AMD) and bioleaching system. [Methods] a co-culture consists of Aph. acidophilum and At. ferrooxidans was separately exposed to four metal ions (Cd2+, Cu2+, Ni2+ and Mg2+) to test its stability. This co-culture was also applied to bioleaching of pyrite and low grade chalcopyrite. [Results] In the metal resistance experiment, heterotrophic bacteria Aph. acidophilum facilitated the ferrous iron oxidation by At. ferrooxidans and improved its efficiency of energy utilization. The maximum tolerant concentration (MTC) of At. ferrooxidans to Cu2+ was improved from 2.0 g/L to 5.0 g/L by Aph. acidophilum, and the cell density of co-culture in 5.0?g/L Cu2+ was almost the same with purely cultured At. ferrooxidans in 2.0 g/L Cu2+. In addition, the MTC of co-cultured At. ferrooxidans to Mg2+ was also improved from 12.0 g/L to 17.0 g/L by Aph. acidophilum. In bioleaching experiment, the pyrite bioleaching efficiency of co-culture increased by 22.70% as compared with that of purely cultured At. ferrooxidans. While in the low grade chalcopyrite bioleaching system with few iron, the bioleaching efficiency of both At. ferrooxidans and its co-culture with Aph. acidophilum were lower than 33%. In the low grade chalcopyrite bioleaching system with pre-added 2 g/L Fe2+, the bioleaching efficiency of At. ferrooxidans and its co-culture with Aph. acidophilum were raised to 41.27% and 52.22%, respectively. [Conclusion] Results in this study demonstrated that At. ferrooxidans and Aph. acidophilum in co-culture could maintain their physiological stability and sustain their ecological function under environmental stress. The bioleaching results suggested that acidophilic heterotrophic bacteria Aph. acidophilum should be applied to the bioleaching system with high iron concentration, in which it could collaborate with iron oxidation bacteria to improve the bioleaching efficiency.