Insect ecdysis triggering hormone (ETH) receptors (ETHRs) are rhodopsin-like G protein-coupled receptors. Upon binding its ligand ETH, ETHR initiates a precisely programed ecdysis behavior series and physiological events. InDrosophila melanogaster, theethrgene produces two functionally distinct splicing isoforms,ethraandethrb. ETH/ETHRA activates eclosion hormone (EH), kinin, crustacean cardioactive peptide (CCAP), and bursicon (burs and pburs) neurons, among others, in a rigid order, to elicit the behavioral sequences and physiological actions for ecdysis at all developmental stages, whereas ETH/ETHRB is required at both pupal and adult ecdysis. However, the role of ETHRB in regulation of molting has not been clarified in any non-drosophila insects. In the present paper, we found that 20-hydroxyecdysone (20E) signaling triggers the expression of bothethraandethrbin a Coleopteran insect pest, the Colorado potato beetleLeptinotarsa decemlineata. RNA interference (RNAi) was performed using double-stranded RNAs (dsRNAs) targeting the common (dsethr) or isoform-specific (dsethra, dsethrb) regions ofethr. RNAi of dsethr, dsethra, or dsethrbby the final-instar larvae arrested larva development. The arrest was not rescued by feeding 20E. All theethradepleted larvae stopped development at prepupae stage; the body cavity was expanded by a large amount of liquid. Comparably, more than 80% of theethrbRNAi larvae developmentally halted at the prepupae stage. The remainingLdethrbhypomorphs became pupae, with blackened wings and highly-expressedburs,pbursand four melanin biosynthesis genes. Therefore, ETHRA and ETHRB play isoform-specific roles in regulation of ecdysis during larva-pupa transition inL. decemlineata.