While it was recently reported that the conduction band of iron minerals can mediate electron transfer between Fe(II) and different Fe(III) lattice sites during Fe(II)-catalyzed mineral transformation, it is unclear whether such a conduction band mediation pathway occurs in the microbial Fe(II) oxidation system under dark and anoxic subsurface conditions. Here, using cytochrome c (c-Cyts) as a model protein of microbial Fe(II) oxidation, the in vitro kinetics and thermodynamics of c-Cyts reduction by Fe(II) were studied. The results showed that the rates of c-Cyts reduction were greatly enhanced in the presence of the semiconducting mineral hematite (Hem, α-Fe2O3). The electrochemical experiments separating Fe(II) and c-Cyts demonstrated that electrons from Fe(II) to the electrode or from the electrode to c-Cyts could directly penetrate hematite, resulting in enhanced current. Independent photochemical and photoluminescence experiments indicated that c-Cyts could be directly reduced by the conduction band electrons of hematite which were generated under light illumination. In c-Cyts+Fe(II)+Hem, the redox potential of Fe(II)-Hem was shifted from -0.15 to -0.18 V and that of c-Cyts+Hem changed slightly from -0.05 to -0.04 V. For the bulk hematite, Mott-Schottky plots illustrated that the flat band was shifted negatively and positively in the presence of Fe(II) and oxidized c-Cyts, respectively, and the surface electron/charge density was higher in the presence of Fe(II)/c-Cyts. As a consequence, the redox gradients from adsorbed Fe(II) to adsorbed c-Cyts allow electron transfer across the conduction band of hematite and facilitate c-Cyts reduction. This mechanistic study on conduction band-mediating electron transfer could help interpret the role of semiconducting minerals in the microbial Fe(II) oxidation process under dark anoxic conditions.