Endometrial-like stromal cells, one of the main components of endometriotic lesions, are an important in vitro model for studying cellular and molecular mechanisms associated with lesion development in endometriosis. However, the short life span of primary endometriotic stromal cells (Ec-ESC) limit their use. Human telomerase reverse transcriptase (hTERT) plasmids can be used to develop immortalized cell lines. Here we aimed to establish an endometriotic stromal cell line by hTERT immortalization. Primary Ec-ESC were obtained from a human ovarian endometriotic cyst. The purity was assessed by morphology and the expression of vimentin, cytokeratin, and human interferon-inducible transmembrane protein 1 (hIFITM1). Cells were infected with hTERT lentiviral vector and selected with hygromycin. hTERT mRNA levels were confirmed by RT-qPCR. Immortalized Ec-ESC (iEc-ESC) were characterized by examining the expression of morphological markers and key genes of interest, TP53, estrogen receptor β (ERβ), progesterone receptor (PR) and steroidogenic factor-1 (SF-1). Karyotyping and in vitro decidualization studies were also performed. Ec-ESCs were positive for vimentin and hIFITM1 and negative for Terms of use and reuse: academic research for non-commercial purposes, see here for full terms.