To determine how UL54-specific siRNA affects virus replication and protection of host cells, we examined virus titer and the activity of the cells at 12 hours, 24 hours, 36 hours, 48 hours, 60 hours and 72 hours after process of RNAi, including: four UL54-specific siRNAs and the positive/negative control siRNAs synthesized in vitro by chemical processes. The Vero cells were transfected with siRNAs using lipofectamine 2000 followed by infection by HSV-II. Our studies reveal that the groups with UL54-specific siRNA decreased significantly in virus titer at 12–24 hours, and only slightly decreased after that; groups with UL54-specific siRNA had higher OD values shown by MTT colorimetric assay than blank cells and survived better; R2 and R4 groups had lower virus titer and better survival than other groups. UL54-specific siRNA can inhibit HSV-II replication, while protecting host cells. There are effective and ineffective siRNA, which were synthesized in accordance with the same principles.