Endometrial cancer (EC) is the third most common gynecologic malignancy in the world, and is considered a chemotherapy poor responding cancer. There are two underlying mechanisms on chemoresistance: the stemness of cancer stem cells (CSCs) and activation of pro-survival autophagy. It was found that autophagy is one of the main factors of cancer stem cell survival, multidrug resistance and maintenance of the homeostasis of cancer stem cells and normal cancer cells. However, the relationship between CSCs and autophagy of EC cells is still unknown. In this study, higher autophagy level was found in endometrial cancer stem cells (ECSCs) and stemness kept in line with autophagy in successive cultured JEC spheres. Autophagy inhibition decreased the properties of CSCs in JEC spheres and enhanced sensitivity of ECSCs to paclitaxel. Besides, it was found that EIG121 exerted dual functions in the regulation of autophagy and stemness not only in normal JEC cells but also JEC obtained CSCs. These findings could be useful for developing targeted therapies for endometrial carcinoma.