Lepidoptera is the second most diverse insect order and includes the most devastating agricultural pests in the world. To ensure the efficient production of crops, people must spray large amounts of chemical pesticides. The use of these chemical insecticides, while killing the insect pests, also caused serious “three R” problems, i.e., resistance, resurgence, and residue. RNA biopesticides use the principle of RNA interference (RNAi) to inhibit the expression of important genes in target organisms, causing the developmental retardation or death of harmful organisms, thereby achieving the purpose of pest control. Compared with traditional pesticides, RNA biopesticides have their own unique features in both mechanism and application development. RNA biopesticides are known as “the third revolution in the history of pesticides” because of their advantages of precision, high efficiency, green, and pollution-free. However, the low RNAi efficiency in Lepidopteran insects severely limits its application in pesticides development and insect pest control. This chapter firstly stated the research and application status of RNAi technology in Lepidopteran insects, and then summarized the factors of dsRNases-dependent and -independent and their effects on RNAi efficiency. Finally, we summarized the recent progress to enhance RNAi efficacy in Lepidopteran insects, including gene target selection, nanoparticles, and genetic engineering of microbes and plants. Hope this chapter can provide a useful reference for the development and supervision of RNA biopesticides in the future.