Abstract: Bacillus subtilis AF0907 with strong inhibitory activity against Fusarium graminearum was isolated from soil. In order to clarify the mechanism of inhibition activity of stain AF0907, the distribution of antagonistic substances was studied by confrontation culture on PDA plates and found to be mainly distributed in cell supernatant. The cell supernatant of strain AF0907 was precipitated by ammonium sulfate at different concentration, and the result showed that 60% ammonium sulfate saturation is the most suitable condition for the antagonistic substance precipitation. The effects of temperature, pH, proteinase K, and chloroform on the inhibition activity of antagonistic substance in AF0907 were then studied. The results showed that antagonistic substance was stable in temperature lower than 60 ℃, but the inhibition activity decreased immediately in temperature higher than 70 ℃. The antagonistic substance was also not stable under acidic or basic conditions. The inhibition activity of the antagonistic substance in AF0907 declined by 60.68%、80.07% after treated with proteinase K and chloroform for one hour in 37 ℃ respectively. According to the antagonistic characterization, the antagonistic substance in AF0907 was identified as protein consequently. The antagonistic protein was then isolated and purified by DEAE-52 column after precipitating by ammonium sulfate and its molecular weight was about 48 kDa detected by SDS-PAGE electrophoresis. The amino acid at N terminal amino acid of the antagonistic protein was sequenced as His-Glu-Phe-Pro-Thr-Tyr-Lys-His-Met-Tyr-Gln-Val-Met-His-Leu by PPSQ-31A protein automated sequencer.