Stem cells play a critical role in endometrial cancer progression. However, the current methodologies used to isolate endometrial cancer stem cells (ECSCs) remain unsatisfactory. The ECSCs were isolated by serumfree suspension cultivation. The stem cells-related genes CD44, CD133, Oct4, Sox2 and Nanog were analyzed, and the biological behaviour of ECSCs was evaluated in vitro and vivo. The results suggest that (i) serumfree suspension cultivation is non-toxic and a convenient way for isolating the ECSCs, and is not limited to specific surface markers; (ii) Ishikawa cells can be used as an effective source of ECSCs, and the obtained ECSCs expressing the pluripotent stem cells markers CD44, CD133, Oct4, Sox2, and Nanog; (iii) ECSCs originated from Ishikawa cells showed an increased ability to invasion and metastasis in vitro, and exhibited a high proliferative capacity and pluripotency in vivo and vitro. These findings indicate that serumfree suspension cultivation is an effective method for isolating ECSCs from Ishikawa cells, and the obtained ECSCs are tumorigenic and display stem cell-like properties. (C) 2017 The Authors. Published by Elsevier Inc.