Spider wasps (Hymenoptera: Pompilidae) are solitary wasps which hunt spiders. Once stung by a pompilid wasp, spiders can stay paralyzed for up to several months, serving as a fresh food source for the wasps’ larvae. Using an integrated proteomic, transcriptomic, and genomic approach, we investigated the venom composition of several pompilid wasp species of the genera Pepsis, Hemipepsis and Heterodontonyx. Their venoms are dominated by peptides derived from a single gene family, characterized by an unusually long repetitive propeptide and diverse short mature peptides. We show that this gene family encodes mature peptides that mimic the arthropod neuropeptide proctolin and the vertebrate hormone bradykinin. Instead of having evolved from endogenous proctolin and bradykinin genes, these peptides evolved de novo. Our study provides insight into the venom composition of pompilid wasps and the molecular mechanisms that have given rise to their venom peptide genes.