Phenoloxidase (PO) plays an important role in arthropod melanization. Previously, a prophenoloxidase (PmproPO1) gene was cloned and characterized from the hemocytes of the black tiger shrimp, Penaeus monodon. In the present study, we report a novel proPO gene (PmproPO2) belonging to the proPO family identified from the P. monodon EST database (http://pmonodon.biotec.or.th). The full-length sequence of PmproPO2 consists of 2513bp encoding a predicted 689 amino acid residues with a calculated molecular mass and pI of 79.21kDa and 6.69, respectively. It is predicted to possess all the expected features of proPO members, including two putative tyrosinase copper-binding motifs with six histidine residues and a thiol ester-like motif, sharing 67% amino acid sequence identity with PmproPO1. Tissue distribution analyses revealed that the two proPO genes are primarily expressed in the hemocyte. Gene silencing of either PmproPO1 or PmproPO2 or both by RNA interference (RNAi) resulted in a significant decrease in the respective endogenous proPO mRNA level in hemocytes and a reduction of total PO enzyme activity by 75, 73 and 88%, respectively. Experimental infection of P. monodon with the pathogenic bacterium, Vibrio harveyi, revealed that PmproPO silenced shrimps were more susceptible to bacterial infection than the control GFP injected shrimps, and suggesting that the two proPOs are important components in the shrimp immune defense.