Myocardial infarction (MI) is the leading cause of death among men and women in the United States. Once a person suffers from a MI, the heart wall will undergo a dynamic and time dependent change, as it goes through the inflammatory phase, proliferative phase, and healing phase. During these phases, the necrotic tissue is removed, and the extracellular matrix (ECM) that holds the cardiomyocytes is altered by an increase in type I collagen, which leads to a scar formation in the infarcted area. The goal of this dissertation is to better understand the role of the cardiac ECM biomechanics in heart physiology, pathophysiology (MI), and regeneration. Three Aims were hence pursued. In Aim 1, we investigated cardiac ECM architecture in intact acellular hearts using diffusion tensor-magnetic resonance imaging (DT-MRI); additionally, we characterized the biomechanical and structural properties of cardiac ECM at different anatomical locations of the left ventricle wall. In Aim 2, we characterized the biomechanical and structural properties of scar ECM during the acute to chronic stages of MI using a rat heart model, in order to better understanding the time course changes in scar ECM biomechanics/microenvironment. In Aim 3, we determined if large mammals (pig heart model) have the capability to fully regenerate a resected piece of the heart apex during the neonatal stage, in which cardiac ECM is still in a developmental phase. The hope is to apply the obtained knowledge in cardiac ECM biomechanics to improve the effectiveness and efficiency of treatments, such as stem cell injection, scar tissue repairing, and regenerative intervention.