Objective To establish a method of neurons and glial cell culture from embryonic Gekko japonicus cerebral cortex. Methods Embryonic (E15) pallium was dissociated and digesting by trypsin. After counting, cells were seeded in culture flask. The glial cells were obtained by using differential adhesion potential combined with successive passage purified methods, and neurons were obtained by using neurobasal medium supplemented with B27. The cells were fixed and analyzed with immunohistochemic assay. Results After tetra_generation, GFAP positive cells were more than 95% in glial cells cultured condition; Neurons grew well in neurobasal medium, and NF and MAP_2 positive signals co_localized on neurons. After cultured for 10 days, the percentage of neurons was more than 95%.Conclusion The methods of isolation, culture and purification for embryonic Gekko japonicus cortical neurons and glial cells were established and it might be a valuable cell model to further investigate