Using American squash (Cucurbita pepo L.) powdery mildew resistant strain F2 and susceptible strain M3 as test materials, we inoculated the powdery mildew physiological race 2US spore suspension in an artificial climate box. The interaction between pumpkin seedling plants and powdery mildew fungus. The reactive oxygen species(ROS) metabolism of leaves and changes in protective enzyme activities after inoculation with powdery mildew were investigated, and the physiological mechanism of pumpkins against powdery mildew was explored. The results showed that: (1) compared with the susceptible strain M3, after inoculation with powdery mildew fungus, the pathogenic fungus on the leaves of the diseaseresistant strain F2 developed slowly and were more difficult to infect the leaves. (2) The H₂O₂ and O₂^-· contents of the diseaseresistant line F2 increased rapidly at the early stage of infection and then gradually decreased, while the H₂O₂ and O₂^-· contents of the susceptible line increased slowly at the initial stage of the disease, and maintained a high level after reaching the maximum, and the MDA content in leaves of susceptible strains was always higher than that of resistant strains. At the same time, through histochemical staining, we found that the leaf coloration of diseaseresistant strains was faster than that of susceptible strains, after which the staining area decreased and tended to a lower level. (3) The activities of antioxidant enzymes CAT, SOD, POD and PAL and PPO in the leaves of diseaseresistant strains F2 and susceptible strains M3 were significantly increased after inoculation with powdery mildew, but the activities and increase of diseaseresistant strains were higher than those of susceptible strains. Studies have found that the development of powdery mildew fungus on the leaves of diseaseresistant American squash for seeds is slow, and it is more difficult to be infected. After the mycelium is formed, the powdery spots on the leaves are smaller; Diseaseresistant strains rely on the increase of active oxygen to resist the invasion of pathogenic fungus at the initial stage of being infected by powdery mildew. As the content of active oxygen increases, diseaseresistant strains rapidly increase their own antioxidant enzyme activities to prevent oxidative stress; compared with susceptible strains, diseaseresistant strains can rapidly increase the activities of PAL and PPO after being infected by pathogenic fungus to resist infection by pathogenic fungus.