Ovarian aging is a pacemaker with multiple organ dysfunction. Recently, stem cells with the ability to generate new oocytes have been identified, which provides the possibility of stem cell therapy for ovarian aging. Several studies have revealed the existence of stem cells in the human postmenopausal ovary. In this study, we describe a new method using magnetic-activated cell sorting combined with differential adhesion to isolate DDX4(+) stem cells from ovaries of postmenopausal women and show that the cells exhibit similar gene expression profiles and growth characteristics with primitive germ cells. Furthermore, the DDX4(+) stem cells could enter the meiosis stage and differentiation into oocytes. The RNA-seq data of the differentiated oocytes shows that mitochondrial metabolism may play an important role in the oogenesis process of the DDX4(+) stem cells. Through using the human ovarian cortical fragments transplantation model, we indicated that the GFP-DDX4(+) stem cells differentiated into some GFP positive oocyte-like structure in vivo. Our study provided a new method for the isolation of DDX4(+) stem cells from the ovaries of postmenopausal women and confirmed the ability of these stem cells to differentiate into oocytes.