Abstract: The objective of this study was to investigate a simple but efficient way to obtain purer ovine endometrial epithelial cells cultured in vitro. In this experiment, two methods were applied as following: one is tissue culturing following trypsin digestion; the another one is collagenase digestion by filtration. Finally, cytokeratin 18 was detected with immunocytochemical staning to evaluate the purity of ovine endometrial epithelial cells collected by these two different ways. The result showed that by method of tissue culturing following trypsin digestion, we could obtain high purity epithelial cells (>95%), which exhibited homogeneous cobblestone morphology and good cell activity. Compared with this method, the collagenase-digesting way by filtration could get lower purity epithelial cells (about 70%), even though the morphology of cells was similar to that of formal one. Collectively, it can be concluded that tissue culturing with trypsin digestion is a simple and efficient way for purifying ovine endometrial epithelial cells, which can be passaged for at least 3-4 generations while they are cultured in vitro.