Regulatory T (Treg) cells may participate in mediating a suppressive microenvironment that blunts successful anti-tumor immunotherapy. Recent studies show that CD8(+) Treg cells might impede effective immune responses to established tumors. However, there is limited research regarding CD8(+) Treg cells in ovarian cancer (OC) patients. Here, we investigated CD8(+) Treg cells in OC patients and their in vitro induction. The immunohistochemistry of tumor-infiltrating lymphocytes revealed a significant correlation between the intratumoral CD8(+) T cells and the forkhead box p3 (Foxp3)(+) cells in the intraepithelial and stromal areas of advanced OC tissues. We examined the expression of Treg markers in CD8(+) T cells from the peripheral blood and fresh tumor tissues of OC patients using flow cytometry. Our results indicated an increase in the CD8(+) Treg cell subsets of OC patients compared with those in patients with benign ovarian tumors and healthy controls, including an increased expression of CD25, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and Foxp3 and decreased CD28 expression. To demonstrate whether the tumor microenvironment could convert CD8(+) effector T cells into suppressor cells, we used an in vitro transwell culturing system. Compared with the CD8(+) T cells cultured alone, the CD8(+) Treg cells induced in vitro by coculture with SK-OV-3/A2780 showed increased CTLA-4 and Foxp3 expression and decreased CD28 expression. In addition, the in vitro-induced CD8(+) Treg cells inhibited naı¨ve CD4(+) T-cell proliferation, which was partially mediated through TGF-β1 and IFN-γ. Our study suggests that CD8(+) Treg cells were increased in OC patients and could be induced in vitro, which may be the way that tumors limit antitumor immunity and evade immune surveillance.