Abstract: RNA silencing (or RNA interference, RNAi) is a conserved mechanism for regulating gene expression in eukaryotes. The discovery of natural trans-kingdom RNAi indicated that small RNAs act as signaling molecules and enable communication between organisms in different kingdoms. The phenomenon and potential mechanisms of trans-kingdom RNAi are among the most exciting research topics. To better understand trans-kingdom RNAi, we review the history of the discovery and elucidation of RNAi mechanisms. Based on canonical RNAi mechanisms, we summarize the major points of divergence around RNAi pathways in the main eukaryotes’ kingdoms, including plants, animals, and fungi. We review the representative incidents associated with the mechanisms and applications of trans-kingdom RNAi in crop protection, and discuss the critical factors that should be considered to develop successful trans-kingdom RNAi-based crop protection strategies.