Objectives: To establish the cultured protocol on Mongolian Gerbil hepatocytes. Method: The hepatocytes were isolated from male Mongolian Gerbil by the methods of tissue digestion and collagenase perfusion. The yield and viability were determined by trypan blue exclusion. The isolated hepatocytes were identified by Periodic acid-Schiff reaction(PAS). The morphology was evaluated with using microscope. Then the medium was repalced with various kinds of cytokines. Results: In sequence of tissue digestion, collagenase perfusion in situ, the hepatocyte’s yield per Mongolian Gerbil was respectively (1.33?.34)?07, (3.97?.15)?07; survival ratio was respectively (29.4?.05)%, (80.3?.56)%. Significant differences were revealed in the yield and viability between the two methods. Numerous glycogenosomes were found in cultured hepatocytes and revealed redness by PAS staining. Significant changes were discovered on morphology after the hepatocytes inoculated in 72 hours. Conclusions: The collagenase perfusion isolation method was economical and high efficiency for separating Mongolian Gerbil hepatocytes. The presence of cytokines in medium could promote the Mongolian Gerbil hepatocyte growth and maintain its function. Successfully establishing the primary cultured protocol of Mongolian Gerbil hepatocytes and it could be used for the reseach on the liver disease and drug exploitation in the future.