Abstract: Bacillus subtilis S44 is a promising plant growth-promoting rhizobacterium and a potent biocontrol agent isolated from cotton rhizosphere. The ituD gene was cloned and identified from S44. A suicide vector containing ituD gene from B. subtilis S44 was recombined into the B. subtilis S44 chromosome to disrupt the ituD gene. Recombination was demonstrated by PCR, antifungal activity and HPLC confirmed the loss of the ituD gene. The growth rate of the recombinant is severely depressed compared with wild type and tetracycline had no effect on the growth rate the mutant strains. Antifungal activity in cell-free extracts of the recombinant has almost no antifungal activity compared with the wild strain. HPLC analysis show that the wild strian produce iturin A at an amount of 89.4 μg mL-1 while the ituD mutant only 3.41 μg mL-1. In pot and plot tests, the control effects of S44 treatment were 80.30 and 72.97%, respectively. The mutant strains S1 and S2 exhibit no control effect of cotton Rhizoctoniosis. These results suggest that the ituD gene was important for iturin A synthesis and biocontrol activity of S44.