Regulatory T cells (T_reg cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T_eff cell) function and gain of suppressive activity by T_reg cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T_reg cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3′ untranslated region. Release of SATB1 from the control of Foxp3 in T_reg cells caused loss of suppressive function, establishment of transcriptional T_eff cell programs and induction of T_eff cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T_reg cell functionality.