The prophenoloxidase (proPO)-activating system in crustaceans and other arthropods is regarded as a constituent of the immune system and plays an important role in defense against pathogens. Hitherto in crustaceans, only one proPO gene per species has been identified. Here we report the identification of a novel proPO-2 (LvproPO-2) from the hemocytes of Litopenaeus vannamei, which shows 72% identity to proPO-1 (LvproPO-1) cloned previously. Northern blotting analysis and quantitative real-time PCR reveal that LvproPO-2 is mainly expressed in the hemocytes, and its expression is down-regulated in shrimp challenged with white spot syndrome virus (WSSV). Western blotting analysis shows that most LvproPO-2/LvPO-2 (L. vannamei phenoloxidase-2) exists in the hemocytes, but not in plasma of L. vannamei. LvproPO-2/LvPO-2 could be detected on the hemocyte surface and the nucleus of hemocytes by indirect immunofluorescence assay (IFA). These findings provide insight into the molecular biological basis for further studying on the defense mechanism of shrimp innate immunity, especially on the proPO-activating system and melanization cascade of shrimp.