Meta-diamides (e.g. broflanilide) and isoxazolines (e.g. fluralaner) are novel insecticides that target the resistant to dieldrin (RDL) subunit of insect γ-aminobutyric acid receptors (GABARs). In this study, we usedin silicoanalysis to identify residues that are critical for the interaction between RDL and these insecticides. Substitution of glycine at the third position (G3’) in the third transmembrane domain (TMD3) with methionine (G3’MTMD3), which is present in vertebrate GABARs, had the strongest effect on fluralaner binding. This was confirmed by expression of RDL from the rice stem borer,Chilo suppressalis(CsRDL) in oocytes of the African clawed frog,Xenopus laevis, where the G3’MTMD3mutation almost abolished the antagonistic action of fluralaner. Subsequently, G3’MTMD3was introduced into theRdlgene of the fruit fly,Drosophila melanogaster, using the CRISPR/Cas9 system. Larvae of heterozygous lines bearing G3’MTMD3did not show significant resistance to avermectin, fipronil, broflanilide, and fluralaner. However, larvae homozygous for G3’MTMD3were highly resistant to broflanilide and fluralaner whilst still being sensitive to fipronil and avermectin. Also, homozygous lines showed severely impaired locomotivity and did not survive to the pupal stage, indicating a significant fitness cost associated with G3’MTMD3. Moreover, the M3’GTMD3mutation in the mouseMus musculusα1β2 GABAR increased sensitivity to fluralaner. Taken together, these results provide convincingin vitroandin vivoevidence for both broflanilide and fluralaner acting on the same amino acid site, as well as insights into potential mechanisms leading to target-site resistance to these insecticides. In addition, our findings could guide further modification of isoxazolines to achieve higher selectivity for the control of insect pests with minimal effects on mammals.