The tricuspid valve is an atrioventricular valve that prevents blood backflow from the right ventricle into the right atrium during ventricular contractions. It is important to study mechanically induced microstructural alterations in the tricuspid valve leaflets, as this aids both in understanding valvular diseases and in the development of new engineered tissue replacements. The structure and composition of the extracellular matrix (ECM) fiber networks are closely tied to an overall biomechanical function of the tricuspid valve. In this study, we conducted experiments and implemented a multiscale modeling approach to predict ECM microstructural changes to tissue-level mechanical responses in a controlled loading environment. In particular, we characterized a sample of a porcine anterior leaflet at a macroscale using a biaxial mechanical testing method. We then generated a three-dimensional finite element model, to which computational representations of corresponding fiber networks were incorporated based on properties of the microstructural architecture obtained from small angle light scattering. Using five different biaxial boundary conditions, we performed iterative simulations to obtain model parameters with an overall R2 value of 0.93. We observed that mechanical loading could markedly alter the underlying ECM architecture. For example, a relatively isotropic fiber network (with an anisotropy index value α of 28%) became noticeably more anisotropic (with an α of 40%) when it underwent mechanical loading. We also observed that the mechanical strain was distributed in a different manner at the ECM/fiber level as compared to the tissue level. The approach presented in this study has the potential to be implemented in pathophysiologically altered biomechanical and structural conditions and to bring insights into the mechanobiology of the tricuspid valve. STATEMENT OF SIGNIFICANCE: Quantifying abnormal cellar/ECM-level deformation of tricuspid valve leaflets subjected to a modified loading environment is of great importance, as it is believed to be linked to valvular remodeling responses. For example, developing surgical procedures or engineered tissue replacements that maintain/mimic ECM-level mechanical homeostasis could lead to more durable outcomes. To quantify leaflet deformation, we built a multiscale framework encompassing the contributions of disorganized ECM components and organized fibers, which can predict the behavior of the tricuspid valve leaflets under physiological loading conditions both at the tissue level and at the ECM level. In addition to future in-depth studies of tricuspid valve pathologies, our model can be used to characterize tissues in other valves of the heart.