A mixed culture of A. ferrooxidans and A. thiooxidans isolated from a coal gangue dump was used to bioleach coal gangue in a column reactor to investigate the leaching of elements. The changes of metal ions (Fe, Mn and Cr) and sulfate in the leaching solution, elemental composition, mineral components and sulfur speciation of the coal gangue before and after bioleaching were analyzed by atomic absorption, anion chromatography, XRF, XRD and XPS. The results show that the mixed culture could promote the release of metal ions in coal gangue, with a leaching concentration of Fe > Mn > Cr. EC and Eh have significantly increased with the increase of metal ion concentrations in the leaching solution. XRF analyses show that the contents of Fe, Mn and S decreased in coal gangue after bioleaching. XRD results suggest that the bioleaching has impacts on minerals in coal gangue, particularly the Fe-containing components. XPS analyses show that sulfur speciation in the raw gangue samples was associated with sulfate, dibenzothiophene and pyrite sulfur. After continuous leaching by the mixed culture, the total sulfur, pyrite sulfur and sulfate sulfur in coal gangue decreased from 2.06% to 1.18%, 0.66% to 0.14% and 1.02% to 0.52%. The desulfurization rates of the pyrite and sulfate were 78.79% and 49.02 %. It is concluded that the mixed culture of these two microorganisms could effectively leach metals from coal gangue coupling with the oxidation of sulfide to sulfate. This study has provided fundamental information as a potential application in the recovery of valuable metals from coal gangue or environmental remediation related to gangue in the future.