CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance by inhibiting the expansion and function of conventional T cells. Treg development and homeostasis are regulated by the Ag receptor, costimulatory receptors such as CD28 and CTLA-4, and cytokines such as IL-2, IL-10, and TGF-β. Here we show that the proportions of Tregs in the spleen and lymph nodes of mice with inactive p110δ PI3K (p110δ D910A/D910A) are reduced despite enhanced Treg selection in the thymus. p110δ D910A/D910A CD4+ CD25+ Foxp3+ Tregs showed attenuated suppressor function in vitro and failed to secrete IL-10. In adoptive transfer experiments, p110δ D910A/D910A T cells failed to protect against experimental colitis. The identification of p110δ as an intracellular signaling protein that regulates the activity of CD4+ CD25+ Foxp3+ Tregs may facilitate the further elucidation of the molecular mechanisms responsible for Treg-mediated suppression.