Objective To compare different regimens of intraperitoneal injection of cyclophosphamide (CTX) to establish a rabbit model of premature ovarian failure (POF), and provide a useful experimental tool for further research of premature ovarian failure. Methods A total of twenty-one 5 -6 months old rabbits were randomly divided into 4 groups. The group A (normal control group) included 3 rabbits without any treatment. The group B (the first model group) included 6 rabbits, received a single intraperitoneal injection of 50 mg/ kg cyclophosphamide. Six rabbits in the group C (the second model group) were injected with 50 mg/ kg cyclophosphamide once daily for 2 days. The group D (the third model group, also n =6) was injected with 50 mg/ kg cyclophosphamide on the first day and then followed by 8 mg/ (kg·d) injection q. d. in the 14 consecutive days. Body weight and ovary weight of the rabbits in each group were measured, and the changes of body weight and the ovary index were analyzed. Morphological changes of the ovarian follicles were observed by HE staining and the numbers of normal and abnormal follicles at different developmental stages were counted and analyzed. Cell apoptosis was analyzed by TUNEL staining and changes in the serum levels of estradiol (E2) were detected by ELISA. Results The body weight of rabbits in both groups B and group C was not significantly changed during the experimental period (P > 0.05). Rabbits in the group D showed a slight growth (P < 0.05) and high mortality. The ovary index in group C was significantly lower than that in the group A (P < 0.05). The ratios of abnormal primordial and primary follicles in groups B and C were significantly increased (P < 0.017), and the ratio of abnormal primordial follicles in the group C was increased more significantly (P < 0.017). However, there was no significant difference in the ratios of abnormal primary follicles between groups B and C (P > 0.017). Among the groups A, B and C, there was no significant difference in the ratios of abnormal secondary and antral follicles (P > 0.05). Apoptosis mainly occurred in granulosa cells of the ovarian follicles. The apoptosis rate of groups B and C was significantly higher than that in the group A (P < 0.05), and the apoptosis rate of group C was higher than that in the group B (P < 0.05). In the group B, the serum E2 level reached the peak value on the 7th day, significantly higher than that on the 35th day (P < 0.05), and then the level was decreased gradually. In the group C, the E2 level was continuously decreased and the level on the last day before drug injection was significantly higher than that at the 35th day (P < 0.05). Conclusions Intraperitoneal injection of 50 mg/ kg cyclophosphamide once daily for 2 days is a most suitable method for the establishment of rabbit model of premature ovarian failure (POF).