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Objective: To culture rabbit endometrial cells by using sex steroids to provide adequate seeding cells for endometrium reconstruction and uterine tissue engineering. Design: Prospective experimental study. Setting: Beijing Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Sciences. Animal(s): New Zealand rabbit and Kunming white strain mice. Intervention(s): Rabbits were primed with pregnant mare serum gonadotropin and hCG. Endometrial cells were cultured with E2 and P4 of different concentrations. The endometrium was reconstructed by using endometrial cells as seeding cells and collagen–basement membrane matrix as scaffolds. Main Outcome Measure(s): Assay with 93-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide, immunofluorescence staining, flow cytometric analysis, hematoxylin and eosin and immunohistochemical staining, and developmental rate of embryos. Result(s): The expression patterns of estrogen receptor and P receptor of rabbit endometrium were different before and after treatment with pregnant mare serum gonadotropin–hCG. One hundred nanomolar E2 with 10 nmol/L P4 facilitated the proliferation of epithelial cells whereas 100 nmol/L P4 facilitated that of stromal cells. The epithelial cells could be stable if cultured for seven or eight passages. Cells in the epithelial layer of the reconstructed endometrium were cytokeratin positive. Some showed columnar morphology akin to the luminal epithelium in vivo. Reconstructed endometrium could improve the developmental rate and quality of one-cell mice embryos. Conclusion(s): Rabbit endometrial cells could be cultured with a long-standing proliferation capability by sex steroids and applied in uterine tissue engineering. Reconstructed endometrium with proliferated endometrial cells was akin to native endometrium in structure and function. [Copyright &y& Elsevier]