Autoimmune inner ear disease is characterized by progressive, bilateral although asymmetric, sensorineural hearing loss. Patients with autoimmune inner ear disease had higher frequencies of interferon-γ-producing T cells than did control subjects tested. Human adipose-derived mesenchymal stem cells (hASCs) were recently found to suppress effector T cells and inflammatory responses and therefore have beneficial effects in various autoimmune diseases. The aim of this study was to examine the immunosuppressive activity of hASCs on autoreactive T cells from the experimental autoimmune hearing loss (EAHL) murine model. Female BALB/c mice underwent β-tubulin immunization to develop EAHL; mice with EAHL were given hASCs or PBS intraperitoneally once a week for 6 consecutive weeks. Auditory brainstem responses were examined over time. The T helper type 1 (Th1)/Th17-mediated autoreactive responses were examined by determining the proliferative response and cytokine profile of splenocytes stimulated with β-tubulin. The frequency of regulatory T (Treg) cells and their suppressive capacity on autoreactive T cells were also determined. Systemic infusion of hASCs significantly improved hearing function and protected hair cells in established EAHL. The hASCs decreased the proliferation of antigen-specific Th1/Th17 cells and induced the production of anti-inflammatory cytokine interleukin-10 in splenocytes. They also induced the generation of antigen-specific CD4+ CD25+ Foxp3+ Treg cells with the capacity to suppress autoantigen-specific T-cell responses. The experiment demonstrated that hASCs are one of the important regulators of immune tolerance with the capacity to suppress effector T cells and to induce the generation of antigen-specific Treg cells.