A cooperative bioleaching (Acidithiobacillus ferriooxidans and Acidithiobacillus thiooxidans) and single bioleaching (Acidithiobacillus ferriooxidans or Acidithiobacillus thiooxidans) of sphalerite were investigated by X-ray diffractometry, energy dispersive spectrography and scanning electron microscopy. The experimental results show that the leaching rate of zinc in the mixed culture is higher than that in pure culture and the sterile control. In these processes, two kinds of bacteria perform different functions and play a cooperative role during leaching of sphalerite. The bioleaching action carried out by Acidithiobacillus ferriooxidans (A. ferriooxidans) is not directly performed through Fe2+ but Fe3+, and its role is to oxidize Fe2+ to Fe 3+ and maintain a high redox potential. Moreover, the addition of an appropriate concentration of ferric iron to the leaching systems is beneficial to zinc dissolution. In the leaching systems without Acidithiobacillus thiooxidans (A. thiooxidans), elemental sulfur layers are formed on mineral surface during the dissolution of zinc and block continuous leaching. Acidithiobacillus thiooxidans, however, eliminate the passivation and cause the bioleaching process to continue in the leaching systems. At the same time, protons from the bacterial oxidization of the elemental sulfur layers also accelerate the leaching of zinc.