Abstract: In this paper, rabbit endometrial cells(epithelial cells and stromal cells) and smooth muscle cells were successfully isolated and cultured, and the ways used in purifying endometrial epithelial cells and stromal cells were discussed. The ways of differential centrifugal speed and differential sticking on the wall were applied to obtain the purified epithelial cells and stromal cells, the way of sieve was chosen to harvest smooth muscle cells, separately. The cells were cultured in the medium that consist of a 1∶1 mixture of DMEM∶F12 media supplemented with 20% FBS, 100 μg/mL insulin, 63.5 nmol/L estrogen, 7.14 nmol/L progesterone,at 37℃ humidified atmosphere of 5% CO2 in air. Immunohistochemistry and immunofluorescence assay were used to identify the three kinds of cells and their purification. Results showed that rabbit endometrial epithelial cells and stromal cells were successfully separated and cultured, the purification of the two kinds of cells reached about 98%. Decidualization was observed in stromal cells. The purification of smooth muscle cells reached about 97%, cells showed lateral-cut growth. The research indicated that endometrial cells and smooth muscle cell could be cultured successfully in vitro; The DMEM/F12(1∶1) culture medium supplemented with 20% FBS,100 μg/mL insulin,63.5 nmol/L estrogen,7.14 nmol/L progesterone and the incubation under condition of 37℃ humidified atmosphere of 5%CO2 were optimal to support the growth of endometrial cells and smooth muscle cells.