Nasonia vitripennis is an important natural enemy of many flies. Pacifastin protease inhibitors (PPIs) play an important role in development and innate immunity of insects. In this study, cDNA sequences of a small pacifastin protease inhibitor in N. vitripennis (NvSPPI) was characterized and its open reading frame (ORF) contains 243bp. Real-time quantitative PCR (RT-qPCR) results revealed that NvSPPI mRNA were detected specifically in the venom apparatus, while they were expressed at low levels in other tissues tested. In the venom apparatus, NvSPPI transcript was highly expressed on the fourth day post eclosion and then declined gradually. The NvSPPI gene was recombinantly expressed utilizing a pGEX-4T-2 vector, and the recombinant products fused with glutathione S-transferase were purified. Inhibition of recombinant GST-NvSPPI to three serine protease inhibitors (trypsin, chymotrypsin, and protease K) were tested and results showed that recombinant NvSPPI could inhibit the activity of trypsin. Meanwhile, we evaluated the influence of the recombinant GST-NvSPPI on the phenoloxidase (PO) activity and prophenoloxidase (PPO) activation of hemolymph from a host pupa, Musca domestica. Results showed PPO activation in host hemolymph was inhibited by recombinant NvSPPI; however, there was no significant inhibition on the PO activity. Our results suggested that NvSPPI could inhibit PPO activation in host hemolymph and trypsin activity in vitro.