Abstract: A. ferrooxidans is an important bioleaching microorganism and the main driving microorganism for the formation of acid mine drainage. To investigate the surface proton adsorption mechanism of A. ferrooxidans, acid-base titration, ProtoFit simulation, electrophoretic mobility determination and Fourier transform attenuated total reflection infrared spectroscopy (ATR-FTIR) were applied to study the effects of different energy sources (S⁰, Fe²⁺, FeS₂) and ionic strength (0.001 mol/L, 0.01 mol/L and 0.1 mol/L NaNO₃) on surface protonation/deprotonation of A. ferrooxidans and the key functional groups involved in the reaction. Results showed that three-site Donnan model could describe the surface complexation of A. ferrooxidans pretty well; The surface properties of A. ferrooxidans were sensitive to both culture energy and ionic strength; A. ferrooxidans was negatively charged in the broad pH range (2-9) studied; The carboxyl group, phosphate group and amide group are the key functional groups in the protonation/deprotonation process. This study indicates important application potential of A. ferrooxidans in the adsorption of heavy metals and has important guiding significance for the elucidation of the surface proton adsorption mechanism of A. ferrooxidans.