BACKGROUNDAphid infestation adversely affects the yield and quality of crops. Rapid reproduction and insecticidal resistance have made controlling aphids in the field challenging. Therefore, the present study investigated the insecticidal property of Penicillium oxalicum (QLhf‐1) and its mechanism of action against aphids, Hyalopterus arundimis Fabricius.RESULTSBioassay revealed that the control efficacy of the spores against aphids (86.30% and 89.05% on the third day and fifth day after infection, respectively) were higher than other components, such as the mycelium. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that QLhf‐1 invaded the aphid cuticle through spores and used the aphid tissues as a nutrient source for growth and reproduction, causing stiffness and atrophy and a final death. Three extracellular enzymes, lipase, protease, and chitinase had a synergistic effect with spores, and they acted together to complete the infection process by degrading the aphid body wall and accelerating the infection process.CONCLUSIONThe newly discovered endophytic penicillin strain P. oxalicum ‘QLhf‐1’ can effectively kill aphids. The results provided strong evidence for the biological control of aphids, and lay a foundation for the development and utilization of QLhf‐1. © 2024 Society of Chemical Industry.