The emergence of imazalil (IMZ) resistance in Penicillium digitatum has become a great threat for controlling citrus green mold. In this paper, we investigated the antifungal efficiency and mechanism of an alkaloid antofine against an IMZ-resistant P. digitatum strain Pdw03. Results showed that antofine exhibited a strong antifungal activity against the mycelial growth of strain Pdw03, with a minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of 1.56 x 10(-3) and 1.25 x 10(-2) g/L, respectively. In vivo application of antofine effectively delayed the disease progress and reduced the incidence of green mold in citrus fruit. The disease incidence of 10 x MFC antofine-treated fruit after 6 days of storage was only 11% +/- 4%, which was significantly lower than that of the control (100% +/- 0%). Antofine treatment altered mycelial morphology of strain Pdw03 without affecting the cell wall integrity. Although the ergosterol contents remained stable, a decrease in the total lipid content induced by lipid peroxidation was observed at 30 min of exposure, indicating disruption of cell membrane permeability of strain Pdw03. In addition, the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) contents were also decreased at 60 min of exposure. These results indicated that antofine inhibited the growth of strain Pdw03 by disrupting cell membrane permeability and impairing energy metabolism induced by oxidative burst. PRACTICAL APPLICATIONS: One of the most economically important postharvest diseases of citrus fruit is green mold caused by Penicillium digitatum. The pathogen is mainly controlled by using imazalil, but the prolonged and extensive application of this chemical fungicide has led to emergence of numerous IMZ-resistant strains among P. digitatum isolates. Consequently, new and safe strategies for controlling citrus green mold caused by IMZ-resistant P. digitatum strains are urgently needed. In this study, an alkaloid antofine effectively inhibited the growth of IMZ-resistant P. digitatum strain Pdw03 and significantly decreased green mold incidence in the affected citrus fruits. Antofine induced membrane lipid peroxidation of Pdw03 mycelia, resulting in damage to the cell membrane and impairment of energy metabolism. Antofine is therefore a potential antifungal agent for the control of green mold, which provide theoretical guidance for the food industry. CI - (c) 2021 Wiley Periodicals LLC.