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Small RNA (sRNA)-mediated RNA interference (RNAi) is a conserved regulatory mechanism for gene expression throughout the domain Eukarya. Recent studies have shown that sRNAs can move between a host and an interacting organism to induce gene silencing in trans, a mechanism termed “Cross-Species RNAi” or, in many cases, “Cross-Kingdom RNAi.” Pathogens and parasites transport sRNAs into host cells during infection and silence host defense genes to suppress immunity, whereas hosts can also deliver their sRNAs into interacting microbes or parasites to suppress infection. Recent studies of different plant and animal hosts and their interacting organisms have unveiled extracellular vesicles (EVs) as vehicles of sRNA exchange in cross-species and cross-kingdom RNAi. The discovery of the pivotal role of sRNAs and EVs in cross-species and cross-kingdom communication offers innovative tools for pathogen and pest control in agriculture and biomedicine.