Regulatory T (T_reg) cells expressing the transcription factor Foxp3 have a pivotal role in maintaining immunological self-tolerance^,,,,; yet, excessive T_reg cell activities suppress anti-tumour immune responses^,,. Compared to the resting T_reg (rT_reg) cell phenotype in secondary lymphoid organs, T_reg cells in non-lymphoid tissues exhibit an activated T_reg (aT_reg) cell phenotype^,,. However, the function of aT_reg cells and whether their generation can be manipulated are largely unexplored. Here we show that the transcription factor Foxo1, previously demonstrated to promote T_reg cell suppression of lymphoproliferative diseases^,, has an unexpected function in inhibiting aT_reg-cell-mediated immune tolerance in mice. We find that aT_reg cells turned over at a slower rate than rT_reg cells, but were not locally maintained in tissues. aT_reg cell differentiation was associated with repression of Foxo1-dependent gene transcription, concomitant with reduced Foxo1 expression, cytoplasmic localization and enhanced phosphorylation at the Akt sites. T_reg-cell-specific expression of an Akt-insensitive Foxo1 mutant prevented downregulation of lymphoid organ homing molecules, and impeded T_reg cell homing to non-lymphoid organs, causing CD8⁺ T-cell-mediated autoimmune diseases. Compared to T_reg cells from healthy tissues, tumour-infiltrating T_reg cells downregulated Foxo1 target genes more substantially. Expression of the Foxo1 mutant at a lower dose was sufficient to deplete tumour-associated T_reg cells, activate effector CD8⁺ T cells, and inhibit tumour growth without inflicting autoimmunity. Thus, Foxo1 inactivation is essential for the migration of aT_reg cells that have a crucial function in suppressing CD8⁺ T-cell responses; and the Foxo signalling pathway in T_reg cells can be titrated to break tumour immune tolerance preferentially.