Objective To establish the approach of isolating, culturing and identifing endometrial stem cells (EnSCs) derived from ectopic lesion of endometriosis patient; and preliminarily examine the biological characteristic of ectopic EnSCs, which provide support for further study on the potential role of ectopic EnSCs in the pathogenesis of endometriosis. Methods The ectopic lesions of endometriosis were harvested from the patients with the informed consent and transferred to lab as soon as possible. The ectopic lesions were minced, digested by collagenase Ⅰ and seeded into cell culture flasks for conventional culture (n=10). Expression of vimentin in ectopic EnSCs was examined by immunofluorescence (n=3). Proliferative capacity of ectopic EnSCs was examined by MTT assay (n=5). Multilineage differentiation potential of ectopic EnSCs was examined by adipogenic and osteogenic differentiation respectively (n=3). Immunophenotype analysis of ectopic EnSCs was determined by flow cytometry (n=3). Production of biological factors in ectopic EnSCs derived conditional medium (n=6) and expression of adhesion molecules on ectopic EnSCs (n=7) were examined by protein assays. Results We successfully isolated EnSCs from ectopic lesions of endometriosis patients, and ectopic EnSCs were positive for vimentin and typical markers of mesenchymal stem cell (CD29, CD73, CD90 and CD105), and negative for the markers of hematopoietic stem cell (CD34 and CD45). The induced ectopic EnSCs showed obvious lipid droplets (adipogenic differentiation) and calcium nodules (osteogenic differentiation). The ectopic EnSCs could secrete high concentration of angiogenic factors ［vascular endothelial growth factor (VEGF), angiotensin (ANG) and platelet-derived growth factor (PDGF)-AA］and angiogenesis associated inflammation cytokines ［interleukin (IL-6)，IL-8 and monocyte chemotactic protein 1(MCP-1)］. Additionally, adhesion molecules analysis demonstrated the high expression of activated leukocyte adhesion molecule（ACLAM）and intercellular cell adhesion molecule-1（ICAM-1）on ectopic EnSCs. Conclusion We successfully establish the procedure of isolating and culturing ectopic EnSCs and demonstrate that ectopic EnSCs is capable of promoting angiogenesis through secreting high concentration of associated biological factors. The above result confirm the existence of EnSCs in ectopic lesions of endometriosis, which not only supports the stem cell based pathogenesis of endometriosis, but also shows the therapeutic potential of taking ectopic EnSCs as promising targets in the treatment of endometriosis.