Background: Pteromalus puparum (Hymenoptera: Pteromalidae) is an endoparasitoid wasp that parasitizes many butterfly species, including a Brassicaceae pest, Pieris rapae (Lepidoptera: Pieridae), the small white cabbage butterfly. P. puparum females inject venom along with their eggs into hosts to ensure successful parasitism. The venom regulates host development and behavior, suppresses host immunity, and influences host metabolism. It has been shown that the venom contains ⊍-amylases, a group of hydrolytic enzymes that act in insect sugar metabolism. So far, three ⊍-amylases have been identified in P. puparum (Pteromalus puparum ⊍-amylases, PpAmys) and the function of PpAmy1 has been reported. However, the functions of PpAmy2 and PpAmy3 remain unknown.Results: We studied the functions of an ⊍-amylase highly expressed in muscle-rich tissues (PpAmy2) and an ⊍-amylase highly expressed in venom apparatus (PpAmy3) using RNAi and GC-TOF-MS techniques. Knockdown of PpAmy3 by RNAi reduced the body length and weight of 1-day old larval offspring while there was no significant effect when PpAmy2 was knocked down. Compared to the control injected with siGFP, many metabolites in P. puparum changed when PpAmy2 was knocked down, while the injection of PpAmy3 recombinant protein into host induced metabolite changes in the P. rapae hemolymph.Conclusion: Our study demonstrated that PpAmy2 acts in metabolism in the muscles of the parasitoid while PpAmy3 could influence the host metabolism and may support the development of parasitic wasp offspring.