AbstractTo balance immunity and tolerance, the endogenous pool of Foxp3⁺ regulatory T (T_reg) cells is tightly controlled, but the underlying mechanisms of this control remain poorly understood. Here we show that the number of T_reg cells is negatively regulated by the kinase Lkb1 in dendritic cells (DCs). Conditional knockout of the Lkb1 gene in DCs leads to excessive T_reg cell expansion in multiple organs and dampens antigen-specific T cell immunity. Lkb1-deficient DCs are capable of enhancing, compared with wild-type DCs, T_reg cell proliferation via cell-cell contact involving the IKK/IKBα-independent activation of the NF-κB/OX40L pathway. Intriguingly, treating wild-type mice with lipopolysaccharide selectively depletes Lkb1 protein in DCs, resulting in T_reg cell expansion and suppressed inflammatory injury upon subsequent challenge. Loss of Lkb1 does not obviously upregulate proinflammatory molecules expression on DCs. We thus identify Lkb1 as a regulatory switch in DCs for controlling T_reg cell homeostasis, immune response and tolerance.