Abstract: Bacillus subtilis B-916 is an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. In this paper, a mutant library was constructed by introducing transposon mini-Tn10 into B-916 genomic DNA. Three mutants which apparent decreased in the inhibition zone to Rhizoctonia solani on plate, were screened from 8 500 mutants compared to B-916. The results showed that the growth curve and biomass were similar to the B-916, but the HPLC analysis results showed that their lipopeptides production were different from B-916. The results of evaluation hemolytic activities of B-916 and mutants showed that mutants’ supernatant hemolytic activities decreased a lot compared to B-916 supernatant. The DNA sequences around the transposon inserted site were also amplified by inverse PCR and sequenced. Multisequence alignments showed that the mutated genes were highly similar to the malonyl coenzyme A transacylaseg gene， lipopeptide synthetase B gene and sensor histidine kinase gene, respectively. All the genes above were relative to synthesis or regulate lipopeptides production. The complete genes above were cloned from B-916 genome by genome walking and designated BacD, BacB and Degs， respectively(submitted to GenBank under accession number FJ192262 and FJ194461). Bioinformatics analysis showed the low similarity lipopeptide synthetase B gene region from B-916 compared to other countpart genes reported previously.