Succinate dehydrogenase (SDH) has been identified as one of the most significant targets for fungicide discovery. Until now, 23 commercial SDHI fungicides have been approved for plant protection since the first launch of carboxin in 1966, and extensively applied to combat destructive plant fungi.In this project, twenty novel pyridine sulfide derivatives containing SDH based heterocyclic amide fungicide were designed, synthesized, and characterized by H-NMR, C-NMR and HRMS. In vitro fungicidal activity experiment, the target compound I-1 displayed excellent inhibitory rates against the common agricultural pathogens with the EC values of 5.2 to 39.8 μg/mL. The in vivo fungicidal activities demonstrated that the compound I-1 could effectively prevent Botrytis cinerea from infecting tomato and cucumber leaves with the preventative rates of 67% and 50%. The mitochondrial membrane potential detection, SDH enzyme assay and the molecular docking simulation revealed that the mechanism of action of the compound I-1 and the relevant interactions with the target enzyme may be similar to those of the control fluopyram.The biological activity screening and validation of mechanism of action indicated that the compound I-1 could be identified as the potential SDH inhibitor for further study. This article is protected by copyright. All rights reserved.