Post-transcriptional gene silencing is an evolutionary conserved mechanism among plants, fungi, insects and animals and engages a sequence specific degradation of RNAs, that is triggered through different pathways by dsRNA molecules. A specific RNAse III, called Dicer cleaves dsRNA molecules into small dsRNAs of 21-24 bp that are the mediators of RNA silencing and, according to their origin, are distinguished into small interfering RNAs (siRNAs) or microRNAs (miRNAs). RNA silencing plays two fundamental roles in the organisms: a key defence role against exogenous RNAs, that counteracts virus infections in animals and in plants, and a gene expression regulatory role, particularly orchestrated during development. As a response to the defensive role of RNA silencing, viruses infecting animals, plants, and fungi have developed special proteins that suppress this process at various steps or allow the virus to evade it. The cross talk between viruses and hosts during infection is not only limited to the evolution and functional development of viral silencing suppressors, but, more interestingly, to the regulatory effects that viruses exert on host genes expression through their activity on endogenous small RNAs to favour their infection process. For all these reasons RNA silencing has now become very useful as an antiviral tool as well as a genetic mean to study gene function in different organisms.