One important aspect of mesenchymal stromal cells (MSCs)-mediated immunomodulation is the recruitment and induction of regulatory T (Treg) cells. However, we do not yet know whether MSCs have similar effects on the other subsets of Treg cells. Herein, we studied the effects of MSCs on CD8⁺CD28⁻ Treg cells and found that the MSCs could not only increase the proportion of CD8⁺CD28⁻ T cells, but also enhance CD8⁺CD28⁻T cells'' ability of hampering naive CD4⁺ T-cell proliferation and activation, decreasing the production of IFN-γ by activated CD4⁺ T cells and inducing the apoptosis of activated CD4⁺ T cells. Mechanistically, the MSCs affected the functions of the CD8⁺CD28⁻ T cells partially through moderate upregulating the expression of IL-10 and FasL. The MSCs had no distinct effect on the shift from CD8⁺CD28⁺ T cells to CD8⁺CD28⁻ T cells, but did increase the proportion of CD8⁺CD28⁻ T cells by reducing their rate of apoptosis. In summary, this study shows that MSCs can enhance the regulatory function of CD8⁺CD28⁻ Treg cells, shedding new light on MSCs-mediated immune regulation.