Gene switches have potential applications for the regulation of transgene expression in plants and animals. Recently, we have developed a two-hybrid ecdysone receptor (EcR) gene switch using chimera 9 [CH9, a chimera between helices 1–8 of Homo sapiens retinoid X receptor (HsRXR) and helices 9–12 of Locusta migratoria RXR (LmRXR)] as a partner for Choristoneura fumiferana EcR (CfEcR). As CH9 includes a region of human RXR, public acceptance of this gene switch for use in genetically modified crops may be an issue. The current studies were conducted to identify an LmRXR mutant that could replace CH9 as a partner for CfEcR. The amino acid identity between LmRXR and HsRXR is fairly high. However, there are a few amino acid residues that are different between these two proteins. LmRXR mutants were produced by changing the amino acids in the helices 1–8 that are different between LmRXR and HsRXR to HsRXR residues. Screening of these mutants in tobacco protoplasts identified a triple mutant, A62S:T81H:V123I (SHILmRXR), that performed as well as CH9. The performance of the EcR gene switch was further improved by optimizing the translational start site (Kozak sequence, AACAATGG) of the transgene. The EcR gene switch containing SHILmRXR and the modified translation start site supported very low background activity in the absence of a ligand and a higher induced activity in the presence of a ligand in tobacco protoplasts, as well as Arabidopsis thaliana transgenic plants. At 16–80 nm methoxyfenozide, the induction of luciferase activity was better than that observed with the CfEcR:CH9 switch.