Regulatory T (T_reg) cells, characterized by expression of the transcription factor forkhead box P3 (Foxp3), maintain immune homeostasis by suppressing self-destructive immune responses^,,,. Foxp3 operates as a late-acting differentiation factor controlling T_reg cell homeostasis and function, whereas the early T_reg-cell-lineage commitment is regulated by the Akt kinase and the forkhead box O (Foxo) family of transcription factors^,,,,. However, whether Foxo proteins act beyond the T_reg-cell-commitment stage to control T_reg cell homeostasis and function remains largely unexplored. Here we show that Foxo1 is a pivotal regulator of T_regcell function. T_reg cells express high amounts of Foxo1 and display reduced T-cell-receptor-induced Akt activation, Foxo1 phosphorylation and Foxo1 nuclear exclusion. Mice with T_reg-cell-specific deletion of Foxo1 develop a fatal inflammatory disorder similar in severity to that seen in Foxp3-deficient mice, but without the loss of T_reg cells. Genome-wide analysis of Foxo1 binding sites reveals ∼300 Foxo1-bound target genes, including the pro-inflammatory cytokine Ifng, that do not seem to be directly regulated by Foxp3. These findings show that the evolutionarily ancient Akt–Foxo1 signalling module controls a novel genetic program indispensable for T_reg cell function.