Regulatory T cells (Treg) prevent the emergence of autoimmune disease. Prototypic natural Treg (nTreg) can be reliably identified by demethylation at the Forkhead-box P3 (FOXP3) locus. To explore the methylation landscape of nTreg we analysed genome-wide methylation in human naïve nTreg (rTreg) and conventional naïve CD4+ T cells (Naïve). We detected 2,315 differentially methylated CpGs between these two cell types, many of which clustered into 127 regions of differential methylation (RDMs). Activation changed the methylation status of 466 CpGs and 18 RDMs in Naïve, but did not alter DNA methylation in rTreg. Gene-set testing of the 127 RDMs showed that promoter methylation and gene expression were reciprocally related. RDMs were enriched for putative FOXP3 binding motifs. Moreover, CpGs within known FOXP3-binding regions in the genome were hypomethylated. In support of the view that methylation limits access of FOXP3 to its DNA targets we showed that increased expression of the immune suppressive receptor TIGIT, which delineated Treg from activated effector T cells, was associated with hypomethylation and FOXP3 binding at the TIGIT locus. We show individuals with autoimmune disease have reduced FOXP3 and TIGIT expression in their rTreg following in vitro activation.