White spot syndrome virus (WSSV) is a major shrimp pathogen causing large economic losses all over the world. So far, however, there is no efficient approach to control this virus. RNA interference (RNAi), which has been applied to silence virus genes in eukaryotic organisms. In this investigation, a specific 21bp short interfering RNA (vp28-siRNA) targeting a major envelope protein gene (vp28) of WSSV was used to induce gene silencing in vivo in Penaeus japonicus shrimp. It was found that the transcription and expression of vp28 gene were silenced by the sequence-specific vp28-siRNA. However, the RNAi effect disappeared or significantly weakened even if one-nucleotide mutation existed in the vp28-siRNA. As revealed by quantitative PCR, the vp28-siRNA caused a significant reduction in viral DNA production of WSSV-infected shrimp. When treated with the vp28-siRNA, WSSV-infected shrimp had a reduced mortality rate. After three injections of the vp28-siRNA, the virus was completely eradicated from WSSV-infected shrimp. These findings suggest that RNAi is capable of silencing sequence-specific genes of WSSV and might constitute a new therapeutic strategy for WSSV infection in shrimp.