The ovarian surface epithelium (OSE) is a single layer of squamous-to-cuboidal epithelial cells that experience repetitive ovulatory rupture and subsequent repair. However, the characteristics of human immortalized ovarian surface epithelial cells (IOSE80) remain elusive. This study aims to determine whether IOSE80 cells have the characteristics of stem cell proliferation and multilineage differentiation and their application in regenerative medicine. IOSE80 cells are sequenced by high-throughput transcriptome analysis, and 5 sets of public data are used to compare the differences between IOSE80 cells and bone marrow mesenchymal stem cells, pluripotent stem cells, and oocytes in transcriptome profiling. The IOSE80 cells present a cobblestone-like monolayer and express the epithelial cell marker KRT18; the stem cell markers IFITM3, ALDH1A1, and VIM; lowly express stem cell marker LGR5 and germ cell markers DDX4 and DAZL. In addition, the GO terms “regulation of stem cell proliferation”, “epithelial cell proliferation”,
etc
., are significantly enriched (
P
<0.05). IOSE80 cells have the potential to act as mesenchymal stem cells to differentiate into adipocytes with lipid droplets, osteoblasts, and chondroblasts
in vitro
. IOSE80 cells express pluripotent stem cell markers, including OCT4, SSEA4, TRA-1-60, and TRA-1-81, and they can be induced into three germ layers
in vitro
. IOSE80 cells also form oocyte-like cells
in vitro
and
in vivo
. In addition, IOSE80 cells exhibit robust proliferation, migration, and ovarian repair functions after
in vivo
transplantation. This study demonstrates that IOSE80 cells have the characteristics of pluripotent/multipotent stem cells, indicating their important role in tissue engineering and regenerative medicine.