Post-transcriptional gene regulation by small RNAs is now established as an important branch of the gene regulatory system. Many different classes of small RNAs have been discovered; among these are short interfering RNAs (siRNAs) and microRNA (miRNAs). Though differences in the processing and function of small RNAs exist between plants and animals, both groups utilize small RNA-mediated gene regulation in response to pathogens. Host encoded miRNAs and siRNAs are generated from viral RNA function in host defense and pathogenic resistance in plants. In animals, miRNAs are key regulators in both immune system development and in immune function. Pathogens, in particular viruses, have evolved mechanisms to usurp the host’s small RNA-mediated regulatory system. Overall, small RNAs are a major component of host defense and immunity in eukaryotes. The goal of this review is to summarize our current knowledge of the involvement of eukaryotic small RNA pathways in host defense and viral pathogenesis.