Mesenchymal stem cells (MSCs), which can be isolated from umbilical cords and induced to differentiate into multiple cell types in vitro, represent an ideal source for cell and gene therapy. MSCs are typically expanded in culture prior to their therapeutic application. However, similar to other types of stem cell, MSCs undergo senescence following a certain number of cell expansion passages in vitro, and eventually stop proliferating. The objective of the present study was to measure the changes that occur over successive passages of MSCs during long‑term in vitro culture, and to detect the effect of aging on MSC morphology, phenotype, proliferation, cell cycle, differentiation, intracellular reactive oxygen species (ROS) levels and gene expression. To understand the importance of oxidative stress in the aging of adult stem cells, the current study established a cell model of H2O2‑induced MSC premature senescence. Analysis of the biological characteristics of human umbilical cord MSCs during replicative and premature senescence revealed the importance of extrinsic factors in the aging of stem cells, particularly ROS. The findings of the present study suggest that cellular senescence, a state of irreversible growth arrest, can be triggered by ROS. Thus, it is important to improve the extrinsic culture environment of MSCs to retain the phenotype of expanded cells and delay the process of senescence prior to their clinical application.