In insect immunity, phenoloxidase (PO) plays an important role in the processes during melanotic encapsulation and phagocytosis. However, uncontrolled PO activity is known to be fatal due to its toxic catalytic products. Thus, the activation of prophenoloxidase (proPO) to PO must be controlled. This study showed that at least three factors were involved in the proPO activation pathway of Spodoptera exigua. Most hemolymph PO activity of S. exigua was detected in hemocytes. The de novo synthesis of proPO was a factor by which PO activity levels were determined in hemolymph, as PO activity was inhibited in a dose-dependent manner by cycloheximide, a eukaryotic protein translation inhibitor. The second factor was derived from serine protease (s) becasue only serine protease inhibitors prevented proPO activation, while other proteases did not. In addition, eicosanoids were implicated in proPO activation, because dexamethasone, an inhibitor of phospholipase $A_2$, inhibited proPO activation. These results indicate that the activation of proPO in S. exigua is controlled at both transcriptional and posttranscriptional levels.