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The endometrial lining of the uterus is essential for women's reproductive health and consists of several different types of epithelial and stromal cells. Although models such as gland-like structures (GLSs) and endometrial assembloids (EnAos) are successfully established, they lack an intact luminal epithelium, which makes it difficult to recapitulate endometrial receptivity. Here, a novel EnAo model (ALI-EnAo) is developed by combining endometrial epithelial cells (EnECs) and stromal cells (EnSCs) and using an improved matrix and air-liquid interface (ALI) culture method. ALI-EnAos exhibit intact EnSCs and glandular and luminal epithelia, which recapitulates human endometrium anatomy, cell composition, hormone-induced menstrual cycle changes, gene expression profiles, and dynamic ciliogenesis. The model suggests that EnSCs, together with the extracellular matrix and ALI culture conditions, contribute to EnAo phenotypes and characteristics reflective of the endometrial menstrual cycle. This enables to transcriptionally define endometrial cell subpopulations. It anticipates that ALI-EnAos will facilitate studies on embryo implantation, and endometrial growth, differentiation, and disease., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)