Simple SummaryInDrosophila melanogasterandTribolium castaneum, dusky-like (Dyl) is related to linking the insect cuticle to the plasma membrane of epidermal cells, regulating the cytoskeleton and deposition of the cuticle. A deficiency in Dyl impairs the formation of embryonic denticles and adult sensory bristles and wing hairs. In a polyphagous beetle,Henosepilachna vigintioctopunctata, we characterizedHvdylusing RNAi. The results showed that the knockdown ofHvdylinhibited the growth of cellular protuberances, inhibited foliage consumption, and affected the survival of the beetles. Our findings indicated that two functions of Dyl inDrosophilaare conserved inH. vigintioctopunctata.AbstractDusky-like (Dyl) is a transmembrane protein containing a zona pellucida domain. Its physiological roles during metamorphosis have been well explored inDrosophila melanogasterand have also been documented inTribolium castaneum. However, Dyl has undergone a functional shift between Diptera and Coleoptera insects. Further investigation of Dyl in other insects will be helpful to further clarify its function in insect growth and development.Henosepilachna vigintioctopunctatais an important Coleoptera that causes enormous economic losses in agriculture in China. In this study, we found that the expression ofHvdylwas detectable in embryos, larvae, prepupae, pupae, and adults. We knocked downHvdylin third- and fourth-instar larvae and pupae with RNA interference (RNAi). RNAi ofHvdylmainly caused two phenotypic defects. Firstly, the growth of epidermal cellular protuberances was suppressed. Injection of dsdyl(double-strandeddusky-likeRNA) at the third-instar larval stage truncated the scoli throughout the thorax and abdomen and shortened the setae on the head capsules and mouthparts of the fourth-instar larvae. Introduction of dsdylat the third- and fourth-instar stages led to misshapen pupal setae. The setae were shortened or became black nodules. Treatment with dsdylat the larval and pupal stages resulted in deformed adults with completely suppressed wing hairs. Moreover, the knockdown ofHvdylat the third-instar stage caused deformed larval mouthparts at the fourth-instar period. As a result, foliage consumption was inhibited, and larval growth was slowed. The results indicate that Dyl is associated with the growth of cellular protuberances throughout development and with the formation of the cuticle inH. vigintioctopunctata.