Regulatory T cells (T_reg cells) are critically involved in maintaining immunological tolerance, but this potent suppression must be 'quenched' to allow the generation of adaptive immune responses. Here we report that sphingosine 1-phosphate (S1P) receptor type 1 (S1P₁) delivers an intrinsic negative signal to restrain the thymic generation, peripheral maintenance and suppressive activity of T_reg cells. Combining loss- and gain-of-function genetic approaches, we found that S1P₁ blocked the differentiation of thymic T_reg precursors and function of mature T_reg cells and affected T_reg cell–mediated immune tolerance. S1P₁ induced selective activation of the Akt-mTOR kinase pathway to impede the development and function of T_reg cells. Dynamic regulation of S1P₁ contributed to lymphocyte priming and immune homeostasis. Thus, by antagonizing T_reg cell–mediated immune suppression, the lipid-activated S1P₁-Akt-mTOR pathway orchestrates adaptive immune responses.