¹College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang, 832003, PR CHINA DOI : 10.9775/kvfd.2017.19055 The aim of this study was to study the effect of steroid hormones on the morphology, growth and proliferation of endometrial cells of laying hens in vitro. Endometrial epithelial and stromal cells were isolated and cultured via type I collagenase digestion, which then were identified by using keratin CK18 or vimentin by immunohistochemistry and treated with steroid hormones, such as E2 and P4, and the growth curves in vitro evaluated by MTT assay. The results showed that endometrial epithelial and stromal cells were successfully isolated and cultured, both of which began to enter the logarithmic phase of growth after 24 h and reached the plateau phase at 7 days after inoculation. Compared to control group, the endometrial epithelial cells were significantly proliferated (P<0.05) by treating with E2 (100 nmol/L) and P4 (100 nmol/L), however the stromal cells were only significantly proliferated (P<0.05) by treating with P4 (100 nmol/L). From these results we can suppose that steroid hormones could positively effect on the proliferation of endometrial cells of laying hens, which would contribute to explore a method to examine the most suitable conditions for endometrial culture in vitro and help to improve further studies on the physiological and functional characteristics of the uterus in the poultry. Keywords : Endometrium, Epithelium cell, Laying hen, Sex hormone, In vitro