Recent applications of RNA interference technology against the white spot syndrome virus (WSSV) were found to be promising and effective antiviral strategies. In the present study, a gene-specific full-length double-stranded RNA (dsRNA) was prepared for the WSSV-VP28 gene using the in vitro transcription method. Challenging experiments were performed by an intramuscular injection of VP28-dsRNA in Marsupenaeus japonicus, followed by WSSV infection after 24h, expecting 95-100% mortality within in 9 days of duration. The results revealed that the protective efficiency of VP28-dsRNA against WSSV infection is more than 85%, 25 days post infection (dpi). Various assays including bioassay, reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, immunohistochemistry (IHC), histology and 4',6-diamidino-2-phenylindole (DAPI) staining were performed to confirm the knockdown of the VP28 gene. No positive signals were observed in the gill and heart tissues of VP28-dsRNA/WSSV-infected shrimp 7dpi by RT-PCR and Western blotting. No infection was observed in the VP28-dsRNA/WSSV-infected group through histology and IHC. DAPI staining of haemocytes revealed no signs of condensation and fragmentation in the VP28-dsRNA-administered group. The unrelated YHVGP116-dsRNA had no significant effect on the expression of the VP28 gene, confirming that the knockdown is specific to VP28-dsRNA. © 2010 Blackwell Publishing Ltd.