Abstract 【Aim】 In our previous studies, the activities of total esterases were found to be significantly increased in the chlorpyrifos, imidacloprid and deltamethrin resistant strains of the small brown planthopper, Laodelphax striatellus. The aim of this study is to identify the carboxylesterase gene causing this increase and to verify its roles in insecticide resistance. 【Methods】 The expression levels of carboxylesterase genes in female adults of the resistant and susceptible strains of L. striatellus were detected by transcriptome sequencing and RT-qPCR. Microinjection was used to establish a transgenic Drosophila melanogaster strain overexpressing the target gene. Topical application method was used to investigate the responses of transgenic D. melanogaster overexpressing the target gene to chlorpyrifos, imidacloprid and deltamethrin, respectively. 【Results】 The expression level of LsCarE1 (GenBank accession no.: HM600723) was upregulated in female adults of the chlorpyrifos, imidacloprid and deltamethrin resistant strains of L. striatellus. Based on the sequence of LsCarE1, codon optimization was performed, and the open reading frame of LsCarE1 was cloned into the transgenic vector pUAST-attB. The transgenic homozygous strain UAS-LsCarE1Optimized was successfully established and then genetically crossed with the GAL4 driver line to obtain a da>LsCarE1Optimized strain that exhibited the overexpression of LsCarE1. The expression level of LsCarE1 in the da>LsCarE1Optimized strain was 257.3 times as high as that in the reference strain UAS-LsCarE1Optimized. The expression of LsCarE1 was not detected in the reference strain da>gal4. Furthermore, the expression levels of the endogenous esterase homologous genes in D. melanogaster were significantly lower than that of LsCarE1. The bioassay results indicated that as compared with the control strain da>gal4, the overexpression strain da>LsCarE1Optimized showed increased tolerance to chlorpyrifos and imidacloprid by 4.19- and 2.46-fold, respectively, but showed no significantly increased tolerance to deltamethrin. 【Conclusion】 The overexpression of LsCarE1 is related to the increased resistance to chlorpyrifos and imidacloprid in L. striatellus.