Abstract: This study compares superovulation efficiency during different stages of the estrus cycle in mice by investigating the pudendum appearance, vaginal epithelial cell type, and the follicular development dynamics, in relation to the estrus cycle. We determined the stages of the estrus cycle by observing the pudendum and making vaginal smears of the Kunming mouse, and ascertaining the follicular development by making paraffin sections. Based on the observation, mice in stages of proestrus, oestrus, metoestrus and dioestrus were superovulated respectively, and their follicular developments and embryo collection results were tracked to ascertain the appropriate estrus cycle stage for superovulation. Our data showed that pudendum appearance and vaginal smears could effectively distinguish different stages of the estrus cycle and that ovarian tissue observation showed that follicular development, corpus luteum formation and luteolysis were regular during the estrus cycle. Likewise, during the estrus cycle of the Kunming mouse, pudendum appearance, vaginal epithelial cell type and follicular development were relational to one another. Superovulating mice in different stages of the estrus cycle showed marked differences both in pregnancy rate and average number of embryos collected. Proestrus was significantly better than metoestrus and dioestrus (P<0.05), and better than oestrus, but the difference between them was not significant (P>0.05). We suggest that pudendum appearance and vaginal smears can be used to distinguish estrus cycle stages and that proestrus is the optimal stage of the estrus cycle for superovulation.