Objective To explore the effects of cyclophosphamide administered by different routes or in different doses on the embryo-fetal development in pregnant rabbits, and to determine the optimal mode of cyclophosphamide administration to induce fetal malformation. Methods Pregnant rabbits were divided into control group C (saline), group Y1 (intravenous injection of 15 mg/kg cyclophosphamide,), group Y2 (subcutaneous injection of low dose cyclophosphamide, 20 mg/kg), and group Y3 (subcutaneous injection of high dose cyclophosphamide, 30 mg/kg). Each rat was administrated according to the corresponding mode once daily on GD10~13. The day of conception was designated as GD0. The pregnant rabbits were sacrificed and dissected on GD28. Then, the number of corpora lutea and implantation, the weight of uterus with contained fetus, and fetal resorption rate were detected, the fetuses were removed and the fetal sex, body length, tail length, the number of live births and stillbirths were recorded, and the appearance of deformities, visceral deformities and skeletal malformations were detected. Results Pregnant rabbit fetuses in the cyclophosphamide intravenous injection group and subcutaneous injection of low dose cyclophosphamide group showed deformities. The appearance malformation rates in the two groups were 30.77% and 95.65%, the skeletal deformity rates were 7.69% and 73.91%, and the visceral abnormality rates were 20.51% and 47.83%, respectively. The fetal resorption rate in the high dose cyclophosphamide subcutaneous injection group was 100%. Conclusions Subcutaneous injection of 20 mg/kg cyclophosphamide to pregnant rabbits at GD10~13 can be used as a positive administrationmethod for rabbit embryo-fetal developmental toxicity test. Thismethod has the advantages of short administration period, easy operation, few fetus resorption, and high rate of fetal malformation, thus, providing the evidence for selection of appropriate model of rabbit embryo-fetal developmental toxicity.