Mesenchymal stem cells (MSCs) are multipotent progenitors capable of self-renewal and differentiation into various cell lineages, making them essential for tissue repair and regenerative medicine. However, their regenerative potential is constrained by replicative senescence, an irreversible growth arrest that occurs after a finite number of cell divisions. In this study, we serially passaged human bone marrow-derived MSCs (bMSCs) and compared young, pre-senescent, and senescent cells. The onset of senescence was accompanied by progressive alterations in mitochondrial dynamics, leading to a decline in mitochondrial membrane potential, and increased reactive oxygen species (ROS) production, alongside a diminished cellular antioxidant capacity. These mitochondrial defects play a role in metabolic reprogramming in senescent bMSCs. Our findings underscore the intricate interplay between ROS, mitochondrial dysfunction, and replicative senescence, offering valuable insights to guide the development of therapeutic strategies for preserving MSC functionality in aging and MSC-based therapies.