This study aimed to evaluate the effect of repeated ovarian stimulation (OS) on the ovarian follicular population and morphology in female mice and its influence on the embryo’s developmental ability, and the profile of the ovarian surface epithelium (OSE). A total of 75 mice were enrolled in this experiment and randomly assigned into three groups: repeated ovarian stimulated group [ =25; receiving 5 IU pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotropin (hCG) at 6 day intervals for 5 cycles]; single ovarian stimulated group ( =25; receiving 5 IU PMSG and hCG for 1 cycle), and control group ( =25; without additional treatment). The follicle number at various stages and the morphologies were recorded respectively in the three groups. The harvested oocytes or embryos, cleavage rate, good quality embryo rate, and blastocyst production rate were counted and calculated, and the proliferations of ovarian surface epithelium were evaluated respectively. In the three groups, the single ovarian stimulation treatment significantly increased the mean number of ovarian oocytes or embryos (39.25±10.77 one-cell embryos/female); on the other hand, repeated gonadotropin stimulation obtained the lowest mean number (5.15± 2.81 eggs/female, <0.01). Repeated ovarian stimulation also tended to decrease normal follicles of primary follicles (66.67%) and secondary follicles (72.86%), and got the lowest cleavage rate (67.47%), lowest good quality embryo rate (2.41%), and lowest blastocyst production rate (0). The OSE cells adjacent to the antral follicles and corpus luteum (CL) in the repeated ovarian stimulated group (81.8%) had a significantly higher proliferation rate than the other groups. The proliferation rate of the OSE in the single ovarian stimulated group (56.4%) was significantly higher than that in the control group (37.5%) ( <0.01). In conclusion, single ovarian stimulation may produce more oocytes/embryos. However, repeated gonadotropin stimulation may have a negative effect on the ovarian follicular quality, the number of mature retrieved oocytes, and the embryo quality, even increasing the chance of ovarian cancer.