CD4⁺ T cells play critical roles in orchestrating adaptive immune responses. Their activation and proliferation are critical steps that occur before they execute their biological functions. Despite the important role of this process, the underlying molecular events are not fully understood. MicroRNAs (miRNAs) have been shown to play important roles in lymphocyte development and function. However, the miRNAs that regulate T-cell differentiation, activation and proliferation are still largely unknown. In our previous study, using a miRNA array, we found that several miRNAs (including miR-202, 33b, 181c, 568 and 576) are differentially expressed between resting and activated CD4⁺ T cells. In this study, we focused on the function of miR-568 during CD4⁺ T-cell activation. We showed that the expression level of miR-568 decreased during the activation of T cells, including Jurkat cells and human peripheral blood CD4⁺ T cells. When Jurkat or human peripheral blood CD4⁺ T cells were transfected with miR-568 mimics, cell activation was significantly inhibited, as shown by the inhibited expression of activation markers such as CD25, CD69 and CD154; decreased IL-2 production; and inhibited cell proliferation. Using software predictions and confirmatory experiments, we demonstrated that nuclear factor of activated T cells 5 (NFAT5) is a target of miR-568. T_reg cells are an important CD4⁺ T-cell subpopulation, so we also evaluated the function of miR-568 in T_reg-cell activation and differentiation. We showed that the miR-568 level decreased, while the NFAT5 protein level increased during CD4⁺CD25⁺ T_reg-cell activation, and the transfection of miR-568 mimics inhibited the NFAT5 expression, inhibited the production of both TGF-β and IL-10 and also inhibited the proliferation of T_reg cells. Our further study showed that over-expression of miR-568 can inhibit T_reg-cell differentiation and can inhibit the suppressive effect of these cells on effector cells. In addition, inhibition of NFAT5 by siRNA-mediated knockdown can inhibit the activation and differentiation of T_reg cells. These findings reveal that miR-568 can inhibit the activation and function of both CD4⁺ T cells and T_reg cells by targeting NFAT5. Since miR-568 plays an important role in both CD4⁺ T cells and T_reg cells, these findings may provide leads for the development of novel treatments for human inflammatory and autoimmune diseases.