[Objective] Bacillus thuringiensis(Bt) D18 is virulent to agricultural pests of Lepidoptera and Coleoptera.This study aims to improve the virulence of D18 by compound mutagenesis.[Methods] After four rounds of atmospheric and room temperature plasma (ARTP) mutagenesis and one round of ARTP-N-methyl-N'-nitro-N-nitrosoguanidine (ARTP-NTG) mutagenesis,highly virulent mutants were screened out by microscopy and virulence bioassay.To reveal the reason for the increase of virulence,we detected the Cry proteins by SDS-PAGE and key cry genes by qRT-PCR.[Results] Two highly virulent mutants,An-L5-1 and An-L5-7 were screened out,compared with the wild strain D18,An-L5-1 and An-L5-7 demonstrated slightly early decline,improved ability of sporulation,80.47% and 88.31% rise of Cry protein expression,and significantly stronger virulence to Plutella xylostella and Mythimna seperata.[Conclusion] The increase in the virulence of An-L5-1 and An-L5-7 was mainly attributed to the significantly up-regulated expression of cry1Aa,cry2Aa,and sigK.