Mesenchymal stem cells (MSCs) are being explored extensively as a promising treatment for autoimmune diseases. We have recently reported that MSCs could ameliorate experimental autoimmune uveoretinitis (EAU) in rats. In this study, we examined further the effects of MSCs on the dynamics of T cell subsets in both eye and spleen and their cytokine production during the course of EAU. We focused on when and where the MSCs had inhibitory effects on T helper type 1 (Th1) and Th17 cells and how long the inhibitory effect lasted, in order to provide more mechanistic evidence for MSCs on the treatment of uveitis. Compared to the control group, administration of MSCs decreased the production of Th1 and Th17 cytokines significantly, while the production of Th2 and regulatory T cell (Treg) cytokines [interleukin (IL)-10 and transforming growth factor (TGF)-β] was elevated during the entire course of EAU. Correspondingly, the dynamic levels of IL-17 in the aqueous humour (AqH) were reduced in MSC-treated rats. Moreover, the ratio of Th17/Treg cells in both spleen and eye was decreased. These results provide powerful evidence that MSCs can regulate negatively both Th1 and Th17 responses and restore the balance of Th17/Tregs in the whole course of EAU, which is important for the regression of the disease.