Abstract: Conditions and characteristics of chicken embryonic cecum epithelial cells (CEC) in subculture were studied in order to provide an in vitro model for the research of E-tenella’s injury mechanism and coccidiostats. Primary chicken embryonic CEC were isolated by digestion combined or step by step with trypsin and EDTA. The digestion method was selected by evaluating the coverage rate of adherent cells. The optimal concentrations of L-GLN and insulin in subculture medium were identified. The CEC were further purified by differential attachment. Characteristics of passage cells were investigated by morphological and SEM observation, immunohistochemistry, histochemistry, PAS, and flow cytometry. Under the conditions of digestion combined with trypsin and EDTA and removal of fibroblasts using differential attachment for 15 min, the CEC grew better in subculture medium with 72.5 mg·L^-1 L-GLN, 0.05 mg·L^-1 insulin, and 5% FBS. The cultured cells had the typical morphological features of epithelium. 24-72 h after passage, the cell purity was more than 95%, and the coverage rate of adherent cells was more than 85%. Results of apoptosis detection showed that high viability was obtained on 5 passages. While the apoptosis rate was significantly increased and coverage rate of adherent cells was decreased after the 6th passage. The subculture conditions were optimized for harvest stable chicken embryonic CEC with large quantity, high viability and purity.