CD4⁺CD25⁺ regulatory T cells play an important role in peripheral tolerance. These cells have been reported to be capable of suppressing the response of CD4⁺CD25⁻ T cells in vitro. The depletion of these cells evokes effective immune responses to tumor cells in vivo. In this study, we demonstrate that CD4⁺CD25⁺ T cells also suppress all subsets of Vα24⁺NKT cells (Vα24⁺CD4⁻CD8⁻ double negative, Vα24⁺CD4⁺, and Vα24⁺CD8⁺) in both proliferation and cytokine production [IFN-γ, interleukin-4 (IL-4), IL-13, and IL-10]. This suppression is mediated by cell-to-cell contact but not by a humoral factor or the inhibition of antigen-presenting cells. Moreover, the cytotoxic activity of Vα24⁺NKT cells against some tumor cell lines is suppressed by CD4⁺CD25⁺ T cells. This finding is important in developing an effective immunotherapy for cancer.