Ovarian cancer is the deadliest gynecological cancer because it has few early symptoms and metastasizes to the surrounding organs at advanced stages. Cancer stem cells (CSCs), a subpopulation of cells with acquired drug resistance, contribute to the recurrence and poor prognosis of ovarian cancer. CD109, a cell surface glycoprotein, has been reported to be a marker of CSCs; however, it remains unclear whether CD109 is secreted by CSCs. In this study, we investigated the amount of CD109 in conditioned media (CM) of CSC populations from ovarian cancer cell lines and patients with ovarian cancer. The CM of sphere-forming CSCs isolated from ovarian cancer cell lines (A2780 and SKOV3) had higher levels of CD109 than those isolated from their adherent cultured parental cells. Furthermore, higher levels of CD109 were detected on the cell surface and in the CM of sphere-forming CSC populations isolated from patient-derived primary ovarian cancer cells. To clarify whether CD109 is localized to the exosomal fraction secreted from CSCs, extracellular vesicles were isolated from the CM by ultracentrifugation. In addition to the CM, the exosomal fraction of ovarian CSCs contained greater levels of CD109 than the parental cells. These results suggest that CD109 is secreted in a soluble or exosomal form from CSCs, and that the measurement of secreted CD109 may be used as a diagnostic or prognostic marker for ovarian cancer.