Chemotherapy is one of the main approach for ovarian cancer. Cancer stem cells (CSCs) escape chemotherapy and lead to chemoresistance. We previously demonstrated that cordycepin (Cd) inhibited metastasis in human ovarian carcinoma cells, the aim of this study is to investigate the effects of Cd on ovarian cancer stemness. TGF-beta was used to induce chemoresistance of chemotherapeutic agent cisplatin in SKOV-3 ovarian cancer cells. After treating with 100 μM of Cd, cell viability, the percentage of cancer stem cells, and the levels of matrix metalloproteinases (MMPs) were decreased in TGF-beta-induced SKOV-3 cells. Treatment of Cd recovered E-cadherin levels and inhibited vimentin levels while TGF-beta treatment significantly increased the expression of vimentin and PGC-1alpha, and decreased E-cadherin levels in SKOV-3 cells, indicating that the action of Cd on cancer stemness may contribute to the regulation of epithelial-mesenchymal transition (EMT). Cd efficiently attenuated chemoresistance caused by TGF-beta in SKOV-3 cancer stem cells to promote the cytotoxicity of cisplatin.