The removal of sulfur in coal before its combustion can reduce its environmental pollution, and microbial desulfurization should have good application prospects. Some species of bacteria have been used in the process of coal desulfurization, and our previous study demonstrated that mixed culture should be more efficient than pure culture by shaking cultivation. In this study, column leaching was investigated, due to its similarity to the industrial heap leaching. The mixed culture consisted of three strains of bacteria, which were Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans respectively. The ratio of the three strains of bacteria was 1: 1: 1. The column (50 cm height, 650 mm Inner Diameter) bioleaching experiments were carried out at ambient temperature. After desulfurization for 18 days, the ultimate desulfurization rate was 28.66% for total sulfur, which was higher than the previous study of J.Cara (with total sulfur desulfurization rate for 24% after 125 days). This study showed that ferric ions should be in proper concentration, and Leptospirillum ferrooxidans might be in lower ratio for reducing the precipitation of jarosite and improving the efficient of coal desulfurization.