The success of pregnancy depends on a well-established fetal-maternal interface, which consisting of endometrial stromal/epithelial cells, immune cells, endothelial cells and trophoblasts. The endometrial gland, which is composed of glandular epithelial cells, is essential for the survival/development of conceptus by secreting and transporting various paracrine factors. We hypothesized that the soluble factors derived from endometrial gland regulate the decidual macrophage polarization and stromal cell functions. A long-term human endometrial organoid culturing system was established by using endometrial glandular tissue. The derived organoids were treated by sex hormones estrogen (E2), progesterone (P4), and/or human chorionic gonadotropin (hCG) to mimic the estrous cycle and early pregnancy environment. The endometrial organoids can be maintained for long-term and functionally respond to E2 and P4. When further stimulated with pregnancy hCG, they acquired characteristics of gestational endometrium as demonstrated by increased glycodelin-A production. Human monocytes were isolated from female blood by immunomagnetic separation and were differentiated into macrophages using macrophage colony-stimulating factor (M-CSF; 50 ng/ml). The inclusion of the E2+P4-treated endometrial organoid secretome during the M-CSF-induced differentiation increased the expression of decidual macrophage marker indoleamine 2, 3-dioxygenase 1 (IDO-1) in the resulting macrophages. The phagocytosis of the differentiated macrophages was inhibited by the secretome of E2+P4+hCG-treated endometrial organoids. An endometrial organoid and stromal cell co-culture model was also established to study the effect of paracrine factors of endometrial glands on decidualization. Our results showed that endometrial organoid secretome suppressed the progestin-cAMP-induced decidualization of the stromal cells. In conclusion, hormonal treatments modulated the secretome of organoids and thereby their actions on endometrial cells.