Human mesenchymal stem cells (hMSCs) have great therapeutic potential due to their high plasticity, immune privileged status and ease of preparation, as well as a lack of ethical barriers to their use. However, their ultimate usefulness is limited by cellular senescence occurring secondary to increased cellular levels of reactive oxygen species (ROS) during their propagation in culture. The underlying molecular mechanisms responsible for this process in hMSCs remain unclear. An antioxidant polyphenol epigallocatechin-3-gallate (EGCG) found in green tea, is known to activate nuclear factor-erythroid 2-related factor 2 (Nrf2), a master transcriptional regulator of antioxidant genes. Herein, we examined the EGCG-mediated antioxidant mechanism in hMSCs exposed to ROS which involves Nrf2 activation. The H2O2-exposed hMSCs showed cellular senescence with significantly increased protein levels of acetyl-p53 and p21 in comparison with the untreated hMSCs, and these effects were prevented by pre-treatment with EGCG. By contrast, in Nrf2-knockdown hMSCs, EGCG lost its antioxidant effect, exhibiting high levels of acetyl-p53 and p21 following EGCG pre-treatment and H2O2 exposure. This indicates that Nrf2 and p53/p21 may be involved in the anti‑senescent effect of EGCG in hMSCs. Taken together, these findings indicate the important role of EGCG in preventing oxidative stress-induced cellular senescence in hMSCs through Nrf2 activation, which has applications for the massive production of more suitable hMSCs for cell-based therapy.