West Nile virus causes fatal encephalitis in humans, mice, and other vertebrates. In the present study, we demonstrate that small interfering RNAs (siRNAs) inhibit West Nile virus replication in vitro. Moreover, the administration of siRNAs to mice by hydrodynamic injection 24 h before challenge with an intraperitoneal inoculum of West Nile virus reduced the viral load and afforded partial protection from lethal infection. These data show the efficacy of the prophylactic use of siRNAs against a viral infection in vivo and suggest new strategies to combat West Nile virus.