Simple SummaryResearch on the composition of egg parasitoid venom is very limited. In this study, we used a combination of transcriptomic and proteomic approaches to identify the protein fractions of the venom in both eupelmid egg parasitoids,Anastatus japonicusandMesocomys trabalae. We identified 3422 up-regulated venom gland genes inM. trabalaeand 3709 inA. japonicusand analyzed their functions comparatively. By proteome sequencing, we identified 956 potential venom proteins in the venom pouch ofM. trabalae, of which 186 were contained in up-regulated genes simultaneously. A total of 766 proteins were detected in the venom ofA. japonicus, of which 128 venom proteins were highly expressed in the venom glands. The results of functional analysis for these identified venom proteins indicated that the venom proteins inM. trabalaeare well known but not inA. japonicus,which may be related to the host range. These results will provide a theoretical basis for studying the function of egg parasitoid venom and its parasitic mechanism.AbstractParasitic wasps are abundant and diverse Hymenoptera insects that lay their eggs inside or on the external surface of the host and inject venom into the host to create a more favorable environment for the larvae to survive and regulate the host’s immunity, metabolism, and development. But research on the composition of egg parasitoid venom is very limited. In this study, we used a combination of transcriptomic and proteomic approaches to identify the protein fractions of the venom in both eupelmid egg parasitoids,Anastatus japonicusandMesocomys trabalae. We identified 3422 up-regulated venom gland genes (UVGs) inM. trabalaeand 3709 inA. japonicusand analyzed their functions comparatively. By proteome sequencing, we identified 956 potential venom proteins in the venom pouch ofM. trabalae, of which 186 were contained in UVGs simultaneously. A total of 766 proteins were detected in the venom ofA. japonicus, of which 128 venom proteins were highly expressed in the venom glands. At the same time, the functional analysis of these identified venom proteins was carried out separately. We found the venom proteins inM. trabalaeare well known but not inA. japonicus,which may be related to the host range. In conclusion, identifying venom proteins in both egg parasitoid species provides a database for studying the function of egg parasitoid venom and its parasitic mechanism.