Venom proteins act in the immunological interactions between parasitoids and their host insects. The effect of venom proteins on host immunity is not fully understood in pupal parasitoids. We identified the functions of a venom protein, calreticulin (PvCRT), in the pupal ectoparasitoid Pachycrepoideus vindemiae. Here, we report that PvCRT features a signal peptide and two conserved "calreticulin" domains. Multiple sequence alignments show that PvCRT shares 83.54% amino acid identity with CRT from both Pteromalus puparum and Nasonia vitripennis, which infers a close relationship among these three species. Using qPCR analysis, we found a lower expression level of PvCRT (0.27-fold) in the venom apparatus compared to the corresponding carcass. Immunohistochemical localization revealed that PvCRT was ubiquitously expressed in venom gland. The expression of the PvCRT gene in Drosophila transgenic lines via the UAS/Gal4 binary expression system reduced the self-encapsulation phenotype of tu(1)Sz1 mutants. Additionally, studies on humoral immunity indicate that PvCRT does not affect the antimicrobial immune responses of the host. This work on an ectoparasitoid will increase our understanding of venom-mediated host-parasitoid interactions.