Emerging evidence indicates that microRNAs control and modulate immunity. MicroRNAs have not been investigated in acquired aplastic anemia, a T cell-mediated immune disease. Analysis of 84 miRNA expression levels in CD4+ and CD8+ T cells of aplastic anemia patients revealed concurrent downregulation of miR-126-3p, miR-145-5p, miR-223-3p, and miR-199a-5p (> 3 fold change (FC), P < .05) in both T cell populations, which were unique in aplastic anemia compared to other hematological disorders. MiR-126-3p and miR-223-3p were downregulated in CD4+ T effector memory cells, and miR-126-3p, miR-145-5p, and miR-223-3p were downregulated in CD8+ T effector memory and terminal effector cells. Successful immunosuppressive therapy was associated with restoration to normal expression levels of miR-126-3p, miR-145-5p, and miR-223-3p (> 2 FC, P < .05). In CD4+ and CD8+ T cells in aplastic anemia patients, MYC and PIK3R2 were upregulated and proved to be targets of miR-145-5p and miR-126-3p, respectively. MiR-126-3p and miR-145-5p knockdown promoted proliferation and increased interferon-γ and granzyme B production in both CD4+ and CD8+ T cells. Our work describes previously unknown regulatory roles of microRNAs in T cell activation in aplastic anemia, which may open a new perspective for development of effective therapy. Clinicaltrials.gov identifier: NCT 01623167.