Regulatory T (Treg) cells are a distinct T-cell lineage characterized by sustained Foxp3 expression and potent suppressor function, but the upstream dominant factors that preserve Treg lineage-specific features are mostly unknown. Here, we show that Lkb1 maintains Treg cell lineage identity by stabilizing Foxp3 expression and enforcing suppressor function. Upon T-cell receptor (TCR) stimulation Lkb1 protein expression is upregulated in Treg cells but not in conventional T cells. Mice with Treg cell-specific deletion of Lkb1 develop a fatal early-onset autoimmune disease, with no Foxp3 expression in most Treg cells. Lkb1 stabilizes Foxp3 expression by preventing STAT4-mediated methylation of the conserved noncoding sequence 2 (CNS2) in the Foxp3 locus. Independent of maintaining Foxp3 expression, Lkb1 programs the expression of a wide spectrum of immunosuppressive genes, through mechanisms involving the augmentation of TGF-β signalling. These findings identify a critical function of Lkb1 in maintaining Treg cell lineage identity.