Penicillium expansum spores in apple juice were treated with contact glow discharge electrolysis (CGDE), and the effect of different parameters on the efficiency of spore inactivation and the related mechanism were investigated. We found that CGDE effectively inactivated spores of P. expansum in the clarified apple juice. CGDE achieved a 3.71 log inactivation of spores in the juice with 1 cm electrode gap, at 520 V, and discharge time for 15 min. CGDE produced hydrogen peroxide in the juice, and the concentration reached 58.1 mg/L after discharged for 30 min. CGDE destroyed the integrity of the cell membrane in the spores, and reduced the activities of superoxide dismutase, catalase, and peroxidase by 68.1%, 81.8%, and 68.0% after discharged for 30 min. In addition, CGDE inhibited mycelial growth, reduced colony diameter by 18.9%, and decreased the lesion diameter by 41.4% in apples surface-inoculated with P. expansum. Taken together, CGDE could inactivate the spores of P. expansum in apple juice by inducing reactive oxygen species accumulation and disrupting the integrity of cell membrane of spores. Industrial relevanceCGDE can effectively inactivate the spores of P. expansum in apple juice. The device of CGDE is composed of a high voltage DC power supply, electrodes and a reaction vessel, which is connected easily with material pipeline, and suitable for handling liquids. Therefore, CGDE can be applicated in treating liquids contaminated with fungal spores in the food industry.