Update information. Background: Cardiac hypertrophy (CH) is the main pathological basis of malignant cadiovascular diseases such as chronic heart failure (CHF). Mitigating and preventing CH is the key strategy for the treatment of ventricular remodeling in CHF. Yi-Xin-Shu capsule (YXS) is highly applied in clinical treatment of CHF in China for decades. However, the mechanism of YXS has not been revealed yet. Method: In the present research, a cardiac hypertrophic model was induced by coarctation of abdominal aorta on rats. To verify the protective effect of YXS against CH, ultrasonic cardiogram was used to evaluate the cardiac function of rats. Hematein Eosin(HE)-staining and transmission electron microscope were used to measure the morphological changes of cardiomyocytes, respectively. Enzyme Linked Immunosorbent Assay (ELISA) was used to detected the myocardial injury biomarkers. Then, the potential targets regulated by YXS were screened out from proteomic analysis and mass spectrometry image analysis. At last, the targets were validated by real-time quantitative (RT-q) PCR, immunofluorescence and western-blotting method. Results: The results showed that YXS improved the cardiac function of CH rats, as well as attenuated the injuries in morphology and subcellular structure of cardiomyocytes. Also, the abnormal elevation of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and cardiac troponin I (CTnI) in CH rats were alleviated by YXS intervention. A core protein-protein interaction network was established on differentially expressed protein (DEP) with proteomics analysis. GATA binding protein 4 (GATA4) was suggested as the key target regulated by YXS. The results of mass spectrometry image analysis indicated that the differential expression and spatial distribution of cardiac proteins in rats. The expression of cell cycle related proteins as histone deacetylase-1(HDAC1) and retinoblastoma (RB) could also be regulated by YXS. Further validative experiments showed that YXS may attenuate CH by regulating RB/ HDAC1/GATA4 signaling pathway. Moreover, the interaction among the potential active components of YXS against CH and targets regulated by this drug was revealed by the compound-target-function network. Conclustions: These data demonstrated that YXS may protect against CH in rats by regulating RB/HDAC1/GATA4 signaling pathway. And the potential active components of YXS against CH were provided.