Impairment of the suppressive function of regulatory T (T-reg) cells has been reported in myasthenia gravis (MG). In this study, cytokine-related mechanisms that may lead to the defect of T-reg were investigated in patients with anti-acetylcholine receptor antibody-positive MG (AChR+MG). Proliferation and cytokine production of responder T (T-resp) cells in response to polyclonal activation were measured in a suppression assay. The effect of interleukin (IL)-21 on suppression was evaluated in vitro in co-culture. IL-21 increased the proliferation of T-resp cells in T-resp/T-reg co-cultures. T-resp cells from patients with MG secreted significantly lower levels of IL-2. In patients with MG, IL-2 levels did not change with the addition of T-reg to cultures, whereas it decreased significantly in controls. In T-resp/T-reg co-cultures, IL-4, IL-6 and IL-10 production increased in the presence of T-reg in patients. Interferon (IFN)-gamma was decreased, whereas IL-17A was increased in both patient and control groups. IL-21 inhibited the secretion of IL-4 in MG and healthy controls (HC), and IL-17A in HC only. The results demonstrated that IL-21 enhances the proliferation of T-resp cells in the presence of T-reg. An effect of IL-21 mainly on T-resp cells through IL-2 is implicated.