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Endometrial epithelial cells play a significant role in the "dialogue" between the embryo and the mother, but in vitro studies to clarify this are hampered by the limited lifespan of primary cells. As such, it is necessary to develop an in vitro model to study endometrial function. Morphological analysis showed that the pEECs were homogeneous, formed characteristic cobblestone monolayers, and expressed the epithelial cell-specific marker, cytokeratin 18. The isolated and purified cells were transfected with a plasmid encoding human telomerase reverse transcriptase (TERT) gene, pCI-neo-TERT, to establish an immortal endometrial epithelial cell line (iEECs). The transfected cells were cultured with G418 and monoclonal cells were selected for expanded culture. Expression of TERT mRNA was detected by RT-qPCR and protein was quantitated by Western blot. TERT expression was stable and continued to be active with no signs of aging. Assays for cell proliferation and apoptosis indicated higher proliferation and cellular activity in iEECs than pEECs. After stimulated by interferon tau (IFN-τ), both iEECs and pEECs showed similar upregulation levels in all the underlying genes. Taken together, these findings demonstrate that iEECs retained the basic morphology and function of pEECs, providing a robust in vitro model for study of the function of ovine endometrial epithelial cells.