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Botrytis cinerea is one of the most destructive fungal pathogens causing tremendous losses in fresh fruit or vegetables. 3-Methylthio-1-propanol (3-MP) is a naturally occurring food-borne sulfide, which is mainly used to increase the flavor in food. However, the potential application of 3-MP in the postharvest phase to manage fruit fungal diseases has not been explored. In this study, the antifungal activity of 3-MP against B. cinerea was evaluated, and the possible mechanism involved was explored. In vitro 3-MP treatment could effectively inhibit the mycelial growth, spore germination, and germ tube elongation of B. cinerea. 3-MP also impaired the spore viability and membrane integrity of B. cinerea as well as increased the leakage of nucleic acids, proteins, and malondialdehyde (MDA) in B. cinerea. In vivo 3-MP fumigation treatment inhibited the infection of B. cinerea on tomato fruits. Also, the fruits with 3-MP fumigation treatment exhibited higher antioxidant enzyme activity, lower MDA content, and a significant delay of induction of the expression of most of the stress-related genes when compared to the control group. Moreover, a cytotoxicity evaluation revealed that 3-MP had no toxicity to normal cells in a certain concentration range. Collectively, our research results will provide evidence for the development of food-borne sulfide 3-MP as a fungicide in food and agriculture and will provide an important reference for the formulation of B. cinerea biocontrol strategies.