Abstract: Three-dimensional uterine tissue was reconstructed in vitro，which was composed of 3 kinds of cells. Rabbit endometrial cells and smooth muscle cells were successfully isolated and cultured in vitro. Smooth muscle cells less than 3 generations ( 1×10⁷ cells/mL) were mixed with the mixture of 3 mg/mL collagen Ⅰ solution derived from mouse tail tendon and Matrigel gel（4∶1)， and were inoculated quickly into self-made moulds， the cellgel mixture solidified for 30 min in 5% CO₂ at 37 ℃. Endometrial stromal cells ( 1×10⁷ cells/mL) were seeded in the mixture by the same ways， the suspension of endometrial epithelial cells was inoculated on the gel when it was solidified; The tissue were cultured in 5% CO₂ at 37 ℃.HE staining and immunohistochemical staining were used to analyze and identify the threedimensional uterine tissue. The results indicated that three-dimensional uterine tissue was successfully reconstructed in vitro， which was composed of three kinds of cells and whose structure was similar to native uterine tissue. HE staining analyses showed that reconstructed uterine tissue was stretched along the stretching direction on 10 d， cells moved freely in extracellular matrix; The epithelial layer showed curving. Immunohistochemical staining revealed that reconstructed uterine tissue distributed three layers of cells; Cells of coiled epithelial layer showed column aspect. Obviously， endometrial cells and smooth muscle cells successfully grow in extracellular matrix; The cultured system of three dimensional uterine was reconstructed in vitro， three-dimensional uterine tissue was composed of three kinds of cells and similar to native uterine tissue. The established model is feasible for research on the mechanism of embryo implantation.