In most clinical applications, human mesenchymal stem cells (hMSCs) are expanded in large scale before their administration. Prolonged culture in vitro results in cellular senescence-associated phenotypes including accumulation of reactive oxygen species (ROS) and decreased cell viabilities. Profiling of stem cell related genes during in vitro expansion revealed that numerous canonical pathways were significantly changed. To determine the effect of selenocysteine (Sec), a rare amino acid found in several antioxidant enzymes, on the replicative senescence in hMSCs, we treated senescent hMSCs with Sec. Supplementation of Sec in the culture medium in late-passage hMSCs reduced ROS levels and improved the survival of hMSCs. In addition, a subset of key antioxidant genes and Sec-containing selenoproteins showed increased mRNA levels after Sec treatment. Furthermore, ROS metabolism and inflammation pathways were predicted to be downregulated. Taken together, our results suggest the antioxidant effects of Sec on replicative senescence of hMSCs.