RNA interference (RNAi) is a natural process involving double-stranded RNA (dsRNA) interfering with the translation of complementary messenger RNAs (mRNAs) into proteins. Insect cell lines have been used extensively to study RNAi mechanisms. Insect cell lines and RNAi are used to identify and study the function of genes involved in various physiological processes. By introducing dsRNA into insect cell lines, target genes are silenced, leading to loss-of-function phenotypes deciphering essential gene functions in insect development, physiology, and immunity. High-throughput RNAi screens were developed using insect cell lines and used to identify key players involved in various cellular and developmental processes such as cell proliferation, apoptosis, differentiation, and signal transduction. Advances in genome sequencing, gene editing, and single-cell sequencing will likely increase the use of insect cell lines and RNAi in functional genomics research. This chapter reviews the use of insect cell lines in RNAi research and the use of insect cell lines and RNAi in functional genomics research.