Forkhead box p3 (FOXP3) is known to program the acquisition of suppressive capacities in CD4+ regulatory T cells (Treg), whereas its role in CD8+ T cells is unknown. The current study investigates whether FOXP3 also acts as a Treg master switch in peripheral blood and tonsillar CD8+ T cells. Single-cell analyses reveal the existence of a FOXP3+ CD8+ population in human tonsils, whereas FOXP3+ CD8+ T cells are rarely detected in peripheral blood. Tonsillar FOXP3+ CD8+ T cells exhibit a Treg phenotype with high CTLA-4 and CD45RO and low CD127 and CD69 expression. Interestingly, the tonsillar FOXP3+ CD8+ T cells are mostly CD25 negative and some cells also express the proinflammatory cytokines TNF-α, IFN-γ, or IL-17A. Particularly, IL-17A-expressing cells are present among FOXP3+ CD8+ T cells. Even though FOXP3 expression is at the detection limit in peripheral blood CD8+ T cells ex vivo, it can be induced in vitro in naive CD8+ T cells by polyclonal stimulation. The induced FOXP3+ CD8+ T cells are predominantly CD25 high and CD28 high and similar to tonsillar cells, they produce high levels of TNF-α, IFN-γ, and granzyme B. However, IL-4 expression is mutually exclusive and IL-17A expression is not detectable. These FOXP3+ CD8+ T cells suppress the proliferation of CD4+ T cells in cocultures, while showing no direct cytotoxic activity. In conclusion, the current study characterizes FOXP3-expressing CD8+ T cells from human tonsils and shows that in vitro activation leads to FOXP3 expression in CD8+ T cells and gain of suppressive activity.