Objectives To investigate the estrus cycle in Mongolian Gerbils, to explore the mechanisms of their regularity, and to optimize the judgment criteria of estrus cycle phases. Methods Vaginal smears were consecutively taken from the gerbils for 18 d, and keratinocyte count was used to observe the vaginal exfoliative cytology under light microscope in 50 female gerbils. The advantages and disadvantages of Wright's staining, HE staining and direct examination under light microscope in distinguishing the four phases of estrus cycle were compared. Results There were three types of estrus cycles is Mongolian Gerbils as stable form, unstable form and pseudopregnancy form. Among these, the stable form accounted for 68.6%. The estrus cycles lasted for (106.3±35.0) hours. Moreover, it could be distinguished into four phases. The proportion of cornified cells in the four phases were: proestrus (13.5±7.8)%, estrus (86.7±9.9)%, metoestrus (27.9±12.8)%, and intermediate stage (3.3±2.8)%. Conclusions Cornified cell count can be used to assess the estrus cycle and each phase correctly. Direct examination under light microscope is ideal for revealing the morphology of vaginal exfoliative cells rapidly. The study of estrus cycle in Mongolian Gerbils may provide a theoretical basis for superovulation, fertilization in vitro, embryo freezing, and biological purification and so on.