Mesenchymal stem cells (MSCs) exhibit a potent immunomodulatory capacity and have been applied to treat diseases such as graft versus host disease and severe autoimmune diseases. However, the mechanism underlying their immunosuppressive effect is not yet completely understood. Here, we investigated the role of the CD73/adenosine pathway in immune modulation by MSCs using a mouse model of experimental autoimmune uveitis (EAU). Moreover, we examined the in vitro modulatory effect of MSCs mediated through the CD73/adenosine pathway in human and mouse T cells. We found that the severity of EAU was significantly attenuated by MSCs; however, most therapeutic effects of MSCs were lost by pretreatment with a CD73 inhibitor. The inhibitory mechanism of MSCs might be contributed by CD73 on MSCs that cooperated with CD39 and CD73 on activated T cells to produce adenosine, resulting in inhibition of T-cell proliferation. Furthermore, MSCs increased the expression of CD73 on CD4(+) T cells, and transforming growth factor-β1 (TGF-β1) was the only tested cytokine that contributed to upregulation of CD73. Hence, our study demonstrates that the CD73/adenosine pathway involves the immunomodulatory function of MSCs in autoimmune responses.