AbstractParasitic wasps inject various virulence factors into the host insects while laying eggs, among which the venom proteins, one of the key players in host insect/parasitoid relationships, act in host cellular and humoral immune regulation to ensure successful development of wasp progeny. Although the investigations into actions of venom proteins are relatively ample in larval parasitoids, their regulatory mechanisms have not been thoroughly understood in pupal parasitoids. Here, we identified a venom protein, Kazal‐type serine protease inhibitor, in the pupal ectoparasitoid Pachycrepoideus vindemiae (PvKazal). Sequence analysis revealed that PvKazal is packed by a signal peptide and a highly conserved “Kazal” domain. Quantitative polymerase chain reaction analysis recorded a higher transcript level of PvKazal in the venom apparatus relative to that in the carcass, and the PvKazal messenger RNA level appeared to reach a peak on day 5 posteclosion. Recombinant PvKazal strongly inhibited the hemolymph melanization of host Drosophila melanogaster. Additionally, the heterologous expression of PvKazal in transgenic Drosophila reduced the crystal cell numbers and blocked the melanization of host pupal hemolymph. Our present work underlying the roles of PvKazal undoubtedly increases the understanding of venom‐mediated host‐parasitoid crosstalk.