Objective  To investigate the molecular protective mechanisms of myocardial hypertrophic preconditioning by mitochondrial quantitative proteomics.   Methods  Fourteen C57BL6/J male mice were randomly divided into sham group(n=6) and cardiac hypertrophy preconditioning group(n=8). The murine model of cardiac hypertrophy preconditioning was established by imposing transverse aortic constriction for 3 days and debanding the aorta for 4 days. Three mice from sham group and four mice from cardiac hypertrophy preconditioning group were randomly selected for proteomic analysis, and the remaining mice were used for functional and morphological experiments. The cardiac function was detected by echocardiography, and mechanical properties of cardiomyocytes were assessed using a SoftEdge Myocam. Cardiac morphology and mitochondrial ultrastructure were detected by pathological sections and transmission electron microscopy. The most significant mitochondrial proteins were screened by label-free quantitative proteomics and analyzed by bioinformatics analysis. Western blotting was used to verify the expression changes.  Results  Compared with the sham group, there were no significant changes in cardiac function and myocardial tissue morphology in the cardiac hypertrophy preconditioning group. However, electron microscopy analyses showed that the density of mitochondrial cristae increased in cardiac hypertrophy preconditioning group. Proteomic analysis screened 20 differentially expressed mitochondrial proteins. Bioinformatics analysis revealed that differentially expressed proteins were mainly related to mitochondrial ribosomal proteins. Western blotting results of key proteins were consistent with proteomic analysis.  Conclusion Myocardial hypertrophic preconditioning can promote the energy metabolism of myocardial mitochondria, which may be related to the transcription, processing and transportation of mitochondrial oxidative phosphorylation complex mediated by mitochondrial ribosomal proteins.