Objective: To investigate the effect of miR-21 knockdown on the proliferation, migration and invasion of endometrial stem cells (EnSCs) derived from ectopic endometrium, and explore the underlying mechanism. Methods: Ectopic endometrial tissues and normal endometrial tissues of endometriosis were collected and EnSCs were isolated respectively. MiR-21 knockdown in EnSCs derived from ectopic endometrium by lentivirus infection. Then, the proliferation, invasion and migration were evaluated by EdU assay and Transwell assay, respectively. EnSCs from ectopic endometrium infected by lentivirus were subcutaneously transplanted into BALB/c nude mice, respectively. The fluorescence intensity of allogeneic cysts were detected by near-infrared fluorescence imaging, then the cysts were excised and the volumes of cysts were measured. The relative protein expression levels of transforming growth factor-β (TGF-β) in EnSCs from ectopic endometrium and normal endometrium were compared by Western blot. The relative protein expression of TGF-β in EnSCs derived from ectopic endometrium infected with lentivirus was detected. Results: The relative expression levels of miR-21 and TGF-β in EnSCs from ectopic endometrium were significantly higher than those in EnSCs from normal endometrium. After miR-21 knockdown, the proliferation, migration and invasion of EnSCs derived from ectopic endometrium were significantly reduced, while TGF-β was decreased. Compared with the negative control group (NC), the fluorescence intensity and volume of cysts formed by EnSCs knockdown of miR-21 were significantly reduced. Conclusion: MiR-21 knockdown inhibits the proliferation, migration and invasion of EnSCs derived from ectopic endometrium, and the mechanism may be related to down-regulating TGF-β expression.