IL-10–competent subset within CD1dhiCD5+ B cells, also known as B10 cells, has been shown to regulate autoimmune diseases. Whether B10 cells can prevent or suppress the development of experimental autoimmune myasthenia gravis (EAMG) has not been studied. In this study, we investigated whether low-dose GM-CSF, which suppresses EAMG, can expand B10 cells in vivo, and whether adoptive transfer of CD1dhiCD5+ B cells would prevent or suppress EAMG. We found that treatment of EAMG mice with low-dose GM-CSF increased the proportion of CD1dhiCD5+ B cells and B10 cells. In vitro coculture studies revealed that CD1dhiCD5+ B cells altered T cell cytokine profile but did not directly inhibit T cell proliferation. In contrast, CD1dhiCD5+ B cells inhibited B cell proliferation and its autoantibody production in an IL-10–dependent manner. Adoptive transfer of CD1dhiCD5+ B cells to mice could prevent disease, as well as suppress EAMG after disease onset. This was associated with downregulation of mature dendritic cell markers and expansion of regulatory T cells resulting in the suppression of acetylcholine receptor–specific T cell and B cell responses. Thus, our data have provided significant insight into the mechanisms underlying the tolerogenic effects of B10 cells in EAMG. These observations suggest that in vivo or in vitro expansion of CD1dhiCD5+ B cells or B10 cells may represent an effective strategy in the treatment of human myasthenia gravis.