RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. In this study, we applied siRNAs targeting the UL39 gene of human herpes simplex virus type 1 (HSV-1), which encodes the large subunit of ribonucleotide reductase, ICP6. Using an ICP6 expression reporter plasmid and an in vitro model of infection, we showed that synthetic siRNA silenced effectively and specifically UL39 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.