Abstract: Objective: To detect and observe the change of mitophagy related protein Bcl-2 19-k Da interacting protein 3 (BNIP3) expression during late cardioprotection of exercise preconditioning, and to determine the relationship between late cardioprotection of exercise preconditioning and myocardial mitophagy. Methods: SD rats were divided into control group (C) , EE group, LEP group, LEP+EE group, and W+LEP+EE group. After establishing of EP animal model, exhaustive exercise on treadmill for inducing myocardial injury. Then the level of c Tn I and hematoxylin basic fuchsin picric acid (HBFP) staining were used to evaluate the degree of myocardial injury and protection. Double immunofluorescence was used to evaluate the transposition of BNIP3 and TOM20. Western blotting method was used to detect change of BNIP3 and LC3. Results: As compared with the group C, the c Tn I level in plasma and mean optical density (MOD) increased significantly, the degree of the transposition of BNIP3 to mitochondria, the level of BNIP3 and LC3II/I increased significantly in group EE;While compared with the group C, there was no significant difference in the c Tn I level, MOD and the level of LC3II/I in group LEP, but the degree of the transposition of BNIP3 to mitochondria and the level of BNIP3 increased significantly. As compared with the group EE, the c Tn I level in plasma and MOD decreased significantly, the degree of the transposition of BNIP3 to mitochondria, the level of BNIP3 and LC3II/I decreased significantly in group LEP+EE. As compared with the group LEP+EE, the c Tn I level in plasma and MOD increased significantly, the degree of the transposition of BNIP3 to mitochondria and the level of BNIP3 increased significantly in group W+LEP+EE. But there was no significant difference in the level of LC3II/I in group W + LEP + EE. Conclusion: Exercise preconditioning can induce mitophagy related protein BNIP3 to increase expression, and the mitophagy induced by BNIP3 participated in late cardioprotection of exercise-induced myocardial injury preconditioning during late cardioprotection of exercise preconditioning.