RNA interference (RNAi) mediated by microRNA (miRNA) is an evolutionarily conserved mechanism of posttranscriptional gene regulation in all eukaryotes, involving in natural antiviral immunity. The RNAase III Drosha is a key component for miRNA maturation. To date, however, the roles of Drosha in virus infection remain to be addressed. In this study, the Drosha was characterized in Marsupenaeus japonicus shrimp. The sequence analysis revealed that the shrimp Drosha gene encoded a 1081-amino-acid peptide, which comprised two tandem ribonuclease III C terminal domains and a double-stranded RNA binding motif. The shrimp Drosha was homologous with those of other animal species. The quantitative RT-PCR analysis revealed that the Drosha gene was highly expressed in lymphoid organ and was significantly up-regulated in response to WSSV challenge, suggesting that the Drosha was involved in the antiviral immunity of shrimp. The results showed that the knock down of Drosha gene led to the defect of miRNA maturation, and subsequent higher virus loads in shrimp. Our study presented that Drosha played important roles in the antiviral defense of shrimp.