RNA silencing is an important antiviral mechanism in plants. Small interfering RNAs (siRNA) or short hairpin RNAs (shRNA) are key stimulators for RNA silencing by acting as executors of viral restriction. Here, we have utilized RNAi technology to suppress potato virus X (PVX) in a transgenic potato cultivar. A stable shRNA of 107 bp directed against a conserved region in the coat protein (CP) gene of PVX was designed with stem and loop sequences derived from a microRNA (miR403; an active regulatory miRNA in potato). The shRNA transgene was directionally cloned in a plant binary vector under the influence of the cauliflower mosaic virus 35S (CaMV35S) constitutive promoter. The pre-shRNA construct was introduced into the potato cultivar Sante through Agrobacterium and transgene insertion was confirmed by testing using PCR (polymerase chain reaction). Upon artificial inoculation of transgenic and non-transgenic potato lines with PVX, variable resistance was revealed through qRT-PCR among the transgenic potato lines. Compared to non-transgenic potato plants, the transgenic potato lines—D5, P3, P9, P14, P21, S11 and S21—showed undetectable levels of CP-PVX mRNA. However, the transgenic lines D4 and P16 exhibited 70% and 75%, respectively, reducing mRNA expression of CP-PVX. The transgenic potato lines remained healthy, with no detectable disease symptoms as compared to the control plants which showed chlorosis and mosaic symptoms. Hence, the expression of virus specific shRNAs is a novel, effective and predictable approach to engineer resistance against PVX in transgenic plants.