Chinese mitten crab（Eriocheir sinensis）hemocyte was treated in vitro with different concentrations of CpG ODN-1670 at 0 μg/mL，5 μg/mL，25 μg/mL，50 μg/mL，75 μg/mL and100 μg/mL in this study. Both intracellular and extracellular relative phenoloxidase （PO） activity （including stimulated activity PO_S and total activity PO_T） were measured，The intra-and extra-celluar PO_S of 25 μg/mL upward treatments and intracellular PO_T of 50 μg/mL upward treatments were significantly enhanced （P < 0.05）；the extracelluar PO_T of all treatments were assumed to be decreased followed the concentration of ODN-1670. These results meant ODN-1670 could induce the transduction of proPO into PO，but it might have some inhibition on the releasing of proPO to outside，which needed further study. Then we monitored the relative expression of prophenoloxidase （proPO） mRNA using real time RT-PCR. The results indicated that 25 μg/mL ODN-1670 could promote relative expression of proPO mRNA significantly （P < 0.05），which suggested new proPO granules synthesize inside of cell. And the PO activity induced by ODN-1670 was enhanced through new synthesis of proPO. In addition，several activators or inhibitors for specific signaling components were applied to treat the exsomalized crab hemocytes cultured in M-199 medium. Detected the intra- or extra-celluar PO activity，we discussed which signal transduction pathway was involved in the ODN-1670 activating proPO system in Chinese mitten crab. The results showed that ODN-1670 could activate the proPO system. It could enhance the PO activity through promoting synthesis of new proPO and transducting of proPO to PO （the activating form）. The signaling pathway of the proPO system activation stimulated by ODN-1670 meight include G-protein mediated PKC way，and was negatively regulated via the tyrosine kinase pathway. Some Toll-Like Receptors might exsit in the cell membrane，and the first step of ODN-1670 active proPO system was to combine with this Toll-Like Receptor protein then activate G-protein mediated pathway.