The effects of energy source (ferrous iron, elemental sulfur and its mixture) on bioleaching of differently pretreated vanadium-bearing shale by Acidithiobacillus ferrooxidans (A. ferrooxidans) were investigated in this paper. The results in the absence of energy source indicated that bioleaching efficiencies of vanadium from raw ore and decarbonized sample were 25.32% and 30.11%, respectively. The positive effects of sulfur as a sole energy source were observed on two types of samples, with vanadium leaching efficiency values of up to 47.86% and 63.32%. However, the bioleaching efficiencies of Fe2+ as a sole energy source were only 36.22% and 25.94%, respectively. Decarbonization promoted the vanadium leaching efficiency of other groups, but growth on only Fe2+ immensely reduced vanadium bioleaching efficiency. The changes of pH, Eh and cell density indicated the relatively higher activities of A. ferrooxidans cultures containing iron in comparison to cultures grown on sulfur as the sole energy source soon after inoculation. A bioleaching mechanism model of differently pretreated vanadium-shale with the culture of Fe2+ as sole energy source was projected. Compared with Fe-containing culture, the addition of sulfur avoided the formation of jarosite, which was more propitious to vanadium extraction. Meanwhile, the sulfur amended cultures achieved higher acidity, which reduced the acid consumption.