The microstructures and mechanical contributions of cardiac elastin in the heart remain poorly understood. In this chapter, we discussed the spatial distribution and network morphology of epicardial, endocardial, interstitial, and Purkinje cell-associated elastin fibers in the porcine left ventricle, as well as their potential biomechanical roles in cardiac function. Using the porcine model, we demonstrated that the epicardial layer, rich in elastin, acts like a prestrained “balloon” that wraps around the heart. We further demonstrated that it is the prestraining of the epicardial layer that provides additional resistance against ventricular diastolic expansion and ventricular wall protection by reducing myocardial stress. Lastly, we verified that the epicardial layer of the healthy human heart has abundant elastin, and the epicardial prestraining phenomenon also exists in the human heart. These discoveries have many implications and potential applications to facilitate biomaterial designs, tissue engineering/3D printing scaffolds, and bioprosthetic interventions for heart disease treatments.