Heart failure is a worldwide cause of mortality and morbidity and is the ultimate ending of a variety of complex diseases. This reflects our incomplete understanding of its underlying molecular mechanisms and furthermore increases the complexity of the disease. To better understand the molecular mechanisms of heart failure, we investigated dynamic proteomic differences between the heart tissue of myocardial infarction rats and the rats in the sham group at days 4, 14, 28, 45 after operation. Using a label-free quantitative proteomic approach based on nanoscale ultra-performance liquid chromatography-ESI-MSE, 133 proteins were identified at the four time points in 8 groups. 13 non-redundant proteins changed dynamically after acute myocardial infarction (AMI) in rat left ventricular (LV) tissue, including cytoskeletal proteins, metabolic enzymes, oxidative stress related proteins and ion channel proteins. The network analysis showed that the differential protein might play an important role in lipid metabolism and hypertrophic cardiomyopathy. The dynamic changes in the expression of beta-actin, alpha B-crystallin (CryAB), heat shock protein 8(HSP8), desmin and L-lactate dehydrogenase B (LDHB) were tested by the western-blot assay, and the results were consistent with the label-free quantitative proteomic results. Correlative analysis indicates that the CryAB and desmin have a better linear relation with heart function (ejection fraction) than cardiac troponin T (cTNT). Our results provide the first experimental evidence of the proteins that are differentially expressed following myocardial infarction, using time-course label-free quantitative proteomics in vivo without ischemia-reperfusion injury or myocardial ischemia. These differential functional proteins (especially CryAB and desmin) have different patterns during the myocardial infarction, which may partially account for the underlying mechanisms involved in cardiac rehabilitation.