AbstractBACKGROUNDRapid evolution of pest resistance has seriously threatened the sustainable use of Bacillus thuringiensis (Bt). The diamondback moth, Plutella xylostella (L.), is the first pest to develop resistance to Bt biopesticides in the open field, which renders it an excellent model to explore the molecular basis of Bt resistance in insects. Our previous midgut transcriptome and RNA‐Seq profiles showed that the P‐glycoprotein gene PxABCB1 was down‐regulated in two Cry1Ac‐resistant P. xylostella strains, suggesting its potential involvement in Cry1Ac resistance in P. xylostella.RESULTSIn this study, the bona fide full‐length cDNA sequence of the PxABCB1 gene was cloned and analyzed, and the expression of the PxABCB1 gene was detected in all tissues and developmental stages, with the highest expression in midgut tissue and the female adult stage. Although no consistent non‐synonymous mutations were identified between the susceptible and resistant strains, PxABCB1 gene expression was remarkably decreased in all resistant strains, and the association was further validated by Cry1Ac selection in the moderately resistant SZ‐R strain. Moreover, knockdown of the PxABCB1 gene expression resulted in significantly reduced larval susceptibility to Cry1Ac toxin in the DBM1Ac‐S strain, and decreased expression of the PxABCB1 gene was tightly linked to Cry1Ac resistance in P. xylostella.CONCLUSIONOur results demonstrated that down‐regulation of the PxABCB1 gene is associated with both laboratory‐selected and field‐evolved Cry1Ac resistance in P. xylostella. This knowledge will be conducive to further elucidating the complicated molecular basis of Bt resistance and developing new insect resistance management tactics. © 2019 Society of Chemical Industry