Prunus necrotic ringspot virus (PNRSV) is a common pollen-borne virus that can infect many Prunus species. While some strains cause serious adverse effects (e.g., rugose mosaic virus), many strains are relatively symptomless in sweet cherry (Prunus avium). However, even symptomless strains can cause adverse reactions in some cherry rootstocks, such as Gisela T (Gi) 7 (P. cerasus x P. canescens). Using interfering RNAs (RNAi) to induce gene silencing, the rootstocks Gi6 (PNRSV-tolerant) and Gi7 (PNRSV-sensitive) were transformed with the RNAi vector pART27-PNRSV containing an inverted repeat (IR) region of PNRSV, using Agrobacterium tumefaciens strain EHA105. This yielded 19 independent transgenic events for Gi6 and three for Gi7. Regenerated plants exhibited a normal phenotype. The presence of the PNRSV-fragment was confirmed by Southern blot. Northern blotting revealed the presence of small interfering RNAs (siRNA) in five of ten transgenic events tested. The transgenic lines were then inoculated with a combination of PNRSV and Prune Dwarf Virus (PDV). After one year, the transgenic Gi7 showed no infection symptoms and was negative for PNRSV in ELISA assays. Non-transgenic Gi7 plants developed serious virus infection symptoms and died. Even though Gi6 is tolerant of PNRSV, inoculation still caused infection, making it a symptomless carrier. In contrast, the inoculated transgenic Gi6 remained negative for PNRSV in ELISA assays. These results indicate RNAi-mediated gene silencing is effective in inducing virus resistance for cherry rootstocks, which may be useful for conferring virus resistance to commercial scions while producing non-genetically modified fruits.