Bradysia difformisis one of the most damaging pests in mushroom production in China. In this study, eightBacillus thuringiensisstrains were analyzed for insecticidal activity inB. difformis. The strain JW-1 showed the highest insecticidal activity againstB. difformislarvae, but did not inhibit the mycelial growth ofPleurotus ostreatusandP. geesteranus. The 16S rRNA gene (1397 bp) andcyt2gene (792 bp) were obtained from strain JW-1. The phylogenetic tree based on 16S rRNA gene and Cyt2 toxin showed that strain JW-1 was a member ofB. thuringiensisand Cyt2 toxin belonged to Cyt2Ba toxin cluster. The Cyt2Ba toxin from strain JW-1 was overexpressed inE. colias a fusion protein and the fusion protein (70 kDa) was purified by Ni-IDA affinity chromatography. The purified Cyt2Ba fusion protein was toxic toB. difformislarvae (LC(50)was 2.25 ng/mL). The identification of Cyt2Ba from strain JW-1 and confirmation of the insecticidal activity of Cyt2Ba inB. difformisprovided a new means of biological control of the important pest in mushroom production.