Human regulatory T cells (T_reg cells) that develop from conventional T cells (T_conv cells) following suboptimal stimulation via the T cell antigen receptor (TCR) (induced T_reg cells (iT_reg cells)) express the transcription factor Foxp3, are suppressive, and display an active proliferative and metabolic state. Here we found that the induction and suppressive function of iT_reg cells tightly depended on glycolysis, which controlled Foxp3 splicing variants containing exon 2 (Foxp3-E2) through the glycolytic enzyme enolase-1. The Foxp3-E2–related suppressive activity of iT_reg cells was altered in human autoimmune diseases, including multiple sclerosis and type 1 diabetes, and was associated with impaired glycolysis and signaling via interleukin 2. This link between glycolysis and Foxp3-E2 variants via enolase-1 shows a previously unknown mechanism for controlling the induction and function of T_reg cells in health and in autoimmunity.