Picornaviruses are a class of RNA viruses with a single-stranded genome in positive orientation. Since the prospects of treatment are limited, we employ RNA interference (RNAi) as an antiviral tool to inhibit different picornaviruses. We identified small interfering RNAs (siRNAs) against the 3D RNA dependent RNA polymerase of coxsackievirus B3 that were capable of efficiently inhibiting the virus. Targeting of the conserved 5' UTR of the virus turned out to be a challenging task since stable structures of this region are detrimental to silencing. We developed a rational strategy to solve this problem and found an siRNA containing locked nucleic acids (LNAs) to possess high antiviral potency. To analyse the mechanism of virus inhibition in more detail, LNAs were incorporated into the siRNA to inactivate either of the siRNA strands. These experiments clearly revealed that only the genomic plus-strand but not the intermediary synthesised minus-strand can be targeted by siRNAs. Furthermore, siRNAs were employed to silence the virus receptor on the host cell and thus prevent viral spread.