The suboptimal outcomes of pelvic organ prolapse (POP) surgery illustrate the demand for improved therapies. However, their development is hampered by the limited knowledge on the cellular pathophysiology of POP. Current investigations, that are limited to tissues and 2D in vitro models, provide highly inconclusive results on how the extracellular matrix (ECM) metabolism and fibroblasts are affected in POP. This study uses a physiologically relevant 3D in vitro model to investigate the cellular pathophysiology of POP by determining the differences between POP and non‐POP fibroblasts on ECM metabolism, proliferation, and fibroblast‐to‐myofibroblast (FMT) transition. This model, based on the synthetic and biomimetic polyisocyanide hydrogel, enables the incorporation of mechanical loading, which simulates the forces exerted on the pelvic floor. Under static conditions, 3D cultured POP fibroblasts are less proliferative, undergo FMT, and exhibit lower collagen and elastin contents compared to non‐POP fibroblasts. However, under mechanical loading, the differences between POP and non‐POP fibroblasts are less pronounced. This study contributes to the development of more comprehensive models that can accurately mimic the POP pathophysiology, which will aid in an enhanced understanding and may contribute to improved therapies in the future. The development of improved surgical therapies for pelvic organ prolapse (POP) is hampered by the limited knowledge on the cellular pathophysiology. Therefore, this study describes a physiologically relevant 3D in vitro model, to which mechanical loading can be applied, that allows to determine the differences between POP and non‐POP fibroblasts on extracellular matrix metabolism, proliferation, and fibroblast‐to‐myofibroblast transition.