Naturally occurring CD4+ regulatory T cells (T R) that express CD25 and the transcription factor FoxP3 play a key role in immune homeostasis, preventing immune pathological responses to self and foreign Ags. CTLA-4 is expressed by a high percentage of these cells, and is often considered as a marker for T R in experimental and clinical analysis. However, it has not yet been proven that CTLA-4 has a direct role in T R function. In this study, using a T cell-mediated colitis model, we demonstrate that anti-CTLA-4 mAb treatment inhibits T R function in vivo via direct effects on CTLA-4-expressing T R, and not via hyperactivation of colitogenic effector T cells. Although anti-CTLA-4 mAb treatment completely inhibits T R function, it does not reduce T R numbers or their homing to the GALT, suggesting the Ab mediates its function by blockade of a signal required for T R activity. In contrast to the striking effect of the Ab, CTLA-4-deficient mice can produce functional T R, suggesting that under some circumstances other immune regulatory mechanisms, including the production of IL-10, are able to compensate for the loss of the CTLA-4-mediated pathway. This study provides direct evidence that CTLA-4 has a specific, nonredundant role in the function of normal T R. This role has to be taken into account when targeting CTLA-4 for therapeutic purposes, as such a strategy will not only boost effector T cell responses, but might also break T R-mediated self-tolerance.