Objective To evaluate the effect of sulforaphane （SFN） on skeletal muscle damage and fatigue in mice induced by exhaustive exercise （EE）.Methods Sixty male Kunming mice were randomly divided into control group （Control）， exhaustive exercise group （EE）， SFN low-dose group （SFN-L， 20 mg/kg/d）， SFN medium-dose group （SFN-M， 40 mg/kg/d） and SFN high-dose group （SFN-H， 60 mg/kg/d）， each group was 12 animals. After total of 4 weeks of administration， the exhaustive exercise was done. The levels of creatine kinase （CK）， lactate （LA）， lactate dehydrogenase （LDH）， malondialdehyde （MDA）， superoxide dismutase （SOD） and catalase （CAT） in each group were determined. Histopathological changes were observed by hematoxylin-eosin staining. The ultrastructure of mitochondria was observed by transmission electron microscope. Energy levels were assessed by measuring citrate synthase （CS）， succinate dehydrogenase （SDH）， and liver and quadriceps glycogen content. RT-qPCR and Western blot were used to detect mRNA and protein expression levels related to mitochondrial autophagy in skeletal muscle.Results Compared with the EE group， the levels of CK， LA， LDH and MDA in the SFN-L， SFN-M and SFN-H groups reduced significantly， while the SOD and CAT activities increased significantly （P<0.05）. SFN significantly reduced skeletal muscle damage in EE mice and mitochondrial morphology was improved. Compared with the EE group， the liver glycogen and muscle glycogen content， CS expression and SDH activity in the SFN-L， SFN-M and SFN-H groups increased significantly （P<0.05）. Compared with the EE group， the protein expression levels of PINK1， Parkin， Lc3-Ⅱ， p62 and ubiquitin in the quadriceps femoris tissue of the SFN-L， SFN-M and SFN-H groups reduced significantly （P<0.05）. Compared with the EE group， the mRNA expression levels of PINK1 and Parkin in the quadriceps femoris tissues of mice in the SFN-L， SFN-M and SFN-H groups reduced significantly （P<0.05）.Conclusion SFN exerts anti-fatigue effects through inhibiting mitochondrial autophagy mediated by PINK1/Parkin signaling pathway.