[Objective] To explore new pathways for biocontrol of anthracnose by investigating the inhibitory effect of Bacillus subtilis C-D6 against appressorium formation of Colletotrichum capsici. [Methods] The antifungal activity of B. subtilis C-D6 against C. capsici was evaluated by dual-culture method. Optimal medium for production of antifungal active substances was determined in shake flask culture. The antifungal proteins were purified by ammonium sulfate precipitation, followed by Sephadex G-75 column chromatography and anion-exchange chromatography. The molecular mass of the purified proteins were determined by SDS-PAGE. [Results] B. subtilis C-D6 showed high antagonistic activity and inhibitory effect of appressorium formation against C. capsici. The optimum medium for the production of antifungal substances of C-D6 strain was YPD and 14 h after inoculation, the culture filtrate had a significant inhibitory effect on appressorium formation of C. capsici. An antifungal protein, with a molecular mass of 32 kD, was purified from culture filtrates of C-D6 strain and the purified protein exhibited obvious inhibitory activity against appressorium formation of C. capsici. [Conclusion] Our results suggest that B. subtilis C-D6 could be a potential bioagent for control of anthracnose caused by C. capsici.