Potted bitter melon was treated with Bacillus velezensis N46 to explore the mechanism of preventing powdery mildew of bitter melon. The prevention mechanism of N46 was studied by detecting the spore germination rate of powdery mildew pathogen (Podosphaera xanthii), changes in leaf defense enzyme activity, accumulation of reactive oxygen species, cell allergic necrosis, lignin accumulation, changes in the expression of disease-resistance related genes and disease-resistance pathways. The control effect of high concentration (6×10⁸ CFU/mL) of B. velezensis N46 on powdery mildew of bitter melon reached 59.05%. In the N46 treatment group, the range and peak value of the activity of defense enzymes POD, CAT and SOD increased significantly, the accumulation of reactive oxygen species (ROS) increased significantly, the rate of anaphylactic necrosis increased significantly, and the rate of lignin accumulation in cell wall was significantly enhanced. N46 had a significant inhibitory effect on the conidial germination of pathogen powdery mildew. The expression levels of JA reactive marker lipoxygenase gene (LOC111018837), SA reactive marker gene (LOC111017362) and peroxidase gene (LOC111021192) associated with allergic reaction and cell wall strengthening were detected in the N46 treated bitter melon. Compared with the control group, the expression levels of the genes increased significantly when bitter melon was infected with the powdery mildew bacteria, and the expression levels of JA reactive marker genes in the treatment group were also higher than those in the control group when there was no contact with pathogenic bacteria. The results indicated that N46 treated bitter melon maintained a certain level of “preactivation” of resistance genes when it was not exposed to pathogenic bacteria, but showed a higher expression level when it was exposed to pathogenic bacteria. Finally, the JA response pathway was inhibited by ibuprofen (IBU), and the control effect of B. velesiensis N46 on powdery mildew of bitter melon was almost disappeared, suggesting that the generation of N46 induced powdery mildew resistance of bitter melon depended on JA pathway. In conclusion, B. velezensis N46 has a good control effect on powdery mildew of bitter melon by inhibiting the conidium germination of megalomycesfoliata and inducing JA pathway dependent resistance of bitter melon. This study would provide a new method for the biological control of powdery mildew of bitter melon and a theoretical basis for the effect of B. velezensis on the control of powdery mildew of bitter melon.