ABSTRACTDespite entomopathogenic fungi being used in various insect pest control, it is recognized that they could replace more chemical insecticides if they were more efficient. We have found that cotton bollwormHelicoverpa armigeraresponded to the infection of entomopathogenic fungusBeauveria bassianaby activating the Toll pathway. Koinobiont wasps also regulate host immunity and development to ensure the survival of their progeny. Previously, venom protein VRF1 was identified inMicroplitis mediator. It entersH. armigerahemocytes, suppresses the expression of antimicrobial peptides (AMPs) by inhibiting the Toll pathway, and prevents parasite offspring from being encapsulated. With this in mind, we thought that it might be feasible to increase the virulence ofB. bassianaby embeddingVRF1into its genome. Compared with that of wild-type (WT)B. bassiana, the median lethal dose (LD50) of the transformant expressing VRF1 (named BbVRF1) decreased approximately 2.36-fold, and the median time to lethality (LT50) was shortened to 84% when infectingH. armigera(a natural host ofM. mediator). The AMP expression level of hemocytes inH. armigerainfected with BbVRF1 strain was significantly downregulated compared to that in the control group infected with the WT. In addition, the LD50of BbVRF1 against the fall armywormSpodoptera frugiperda(an unnatural host ofM. mediator) was decreased 3.45-fold and the LT50was shortened to 73%, showing a greater virulence. Our research indicated that BbVRF1, an engineered strain ofB. bassiana, has greater efficacy against pest insects both within and outside its host range (M. mediator), expanding the utilization of parasitoid wasp virulence effectors.IMPORTANCEMycoinsecticides are essential for the development of integrated pest management as substitutes to chemical insecticides, but their usage is limited by their inferior virulence. Thus, genetically engineered bioinsecticides, including recombinant entomopathogenic fungi, have been regarded as a breakthrough to rapidly control pests. Deep knowledge of parasitoid wasps allows us to take advantage of this natural enemy of pest insects beyond raising them for field release. Our transformant BbVRF1 (Beauveria bassianaintegrated with a venom protein VRF1 fromMicroplitis mediator) showed a higher virulence inH. armigeraandS. frugiperda, demonstrating its potential for managing natural or unnatural hosts ofM. mediator. This result provides a new strategy regarding which venom protein of parasitoid wasps can become part of the arsenal with which to equip entomopathogenic fungi. Utilizing parasitoid wasps with this approach could easily overcome the difficulties of artificial culture and enhance the virulence of other biocontrol agents.