In this paper, the mechanism of mixed bacteria (Fe²⁺ grown or pyrrhotite grown) leaching of pyrrhotite was investigated through the analysis of adsorption quantity, electrokinetic potential, contact angle, shock-flask bioleaching and leaching residue of mixed bacteria cultured with different materials. The results showed that pyrrhotite-grown mixed bacteria have stronger adsorptivity and biological oxidation ability; absorption of bacteria caused the isoelectric point of pyrrhotiteto shift towards the isoelectric point of bacteris, in which the isoelectric point shifted more for minerals treated with grown mixed bacteria than did with the other bacteria. Acid dissolution was in a dominant position at the preliminary stage of pyrrhotite leaching and bioleaching was gradually taking part into work as the mixed bacteria adsorbed. Throughout the process, relative enrichment of iron element will happen in the system, which resulted in increasing of electrokinetic potential of minerals. Meanwhile, iron element easily produced hydrophilic iron precipitates on surfaces of pyrrhotite, which caused the increase of the hydrophilicity and the decrease of contact angle. Bacterial leaching was indirectly influencing the pyrrhotite leaching, since the minerals were oxidized by Fe³⁺, which was produced by bacteria leaching in solution.