Prophenoloxidase (proPO) is the terminal component of the proPO activating system, a defense and recognition system in crustaceans and insects. It can be activated to generate the active enzyme phenoloxidase (PO) by a proteinase cascade and some additional factors. PO can oxidize phenols to quinones that can then polymerize non-enzymatically to melanin. Recently, molecular biology studies of proPO, such as gene expression, structure analysis, and real time expression, have made great progress. However, these studies focused mainly on shrimps and crabs. To better understand the phylogenetic relations and characteristics of the proPOs gene among crustaceans, it is necessary to study crustaceans other than shrimp and crab. Oratosquilla oratoria belongs to order Stomatopoda in phylum Crustacea. O. oratoria is found on muddy bottoms in the coastal waters of Russia, China, Korea, Japan, Vietnam, and Australia and is exploited commercially in many regions. Its natural resources have degraded by degrees since fishing intensification increased and the ecological environment of inshore areas deteriorated. Artificial breeding of O. oratoria is now practiced successfully. However, just as the sustainability and development of shrimp aquaculture is increasingly threatened by significant ecological and pathological problems, it is likely that O. oratoria aquaculture will develop similar severe issues. To boost the healthy development of O. oratoria aquaculture, much more attention should be paid to the PO enzyme which plays an important part in the immune system of crustaceans. Research on the molecular structure and tissue expression of the proPO gene may give us a more thorough understanding of the immunologic mechanisms in O. oratoria. In this paper, the cDNA of the O. oratoria proPO (O-proPO) gene was cloned using reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The full length cDNA of O-proPO consisted of 2, 436 base pairs (bp) with a 2, 142 bp open reading frame (ORF), which can encode a 713 amino acid protein with a predicted molecular mass of 82, 446 Daltons and an estimated pI of 8.78. The nucleotide sequence of O-proPO showed 82%, 76%, 76%, 72%, 70% and the deduced amino acid sequence of O-proPO showed 51%, 51%, 48%, 51%, and 50% homology with the corresponding sequences from Penaeus monodon, Litopenaeus vannamei, Macrobrachium rosenbergii, Penaeus semisulcatus and Marsupenaeus japonicas respectively. Sequence analysis indicated that three tandem hemocyanin domains (hemocyanin-N domains, hemocyanin-C domains and hemocyanin-M domains) were present in the O-proPO amino acid sequence, confirming that O-proPO is a member of the proPO family. The hemocyanin-M domains contained two conserved copper-binding sites surrounded by six histidine residues that are highly conserved in other crustacean species. Five polyadenylation signal sites were identified in the nucleotide sequence of O-proPO, suggesting that multiple shearing may occur at the 3' end. This phenomenon has rarely been reported in crustaceans. Several immune modulatory sites were also detected in the amino acid sequence of O-proPO; they include a putative cleavage site for zymogen activation by the proPO-activating enzyme (ppA), five predicted N-linked glycosylation sites and an α₂-macroglobulin site, all of which might play a critical role in the immunologic process. The sequence of the α₂-macroglobulin site, ACGWPQHV, was found to be conserved when the O-proPO amino acid sequence was compared with proPO in other crustacean species. Phylogenetic analysis revealed that O-proPO and proPO of decapod species formed two subgroups on the same branch of the tree. These subgroups then clustered with proPO from Artemia franciscana. This finding is consistent with the taxonomic status of these species. Tissue expression of the proPO gene was carried out in antenna, eyestalk, ovarian, intestine, muscle and hemolymph of O. oratoria by semi-quantitative RT-PCR analysis. ProPO expression was detected in hemolymph and intestine, with the higher expression level in hemolymph.