MicroRNAs (miRNAs) are important to maintain immune tolerance. To explore their pathogenic involvement in human autoimmune disease, we performed expression profiling of T cell subsets from healthy individuals and those at high-risk of type 1 diabetes (pre-T1D). Differentially expressed miRNAs were detected in each T cell subsets compared to naïve CD4+ T cells and between healthy and pre-T1D individuals, for example increased expression of miR-26a in activated natural regulatory T cells was associated with a reduction in the blood of EZH2 mRNA, which encodes the catalytic subunit of the polycomb repressive complex 2, in pre-T1D individuals. EZH2 was shown to be required for nTreg survival and for regulating the IL-2 receptor alpha chain (CD25) expression. Our findings suggest that alterations in the expression of specific miRNAs may contribute to impaired Treg function in autoimmune disease-prone individuals.