Objective To investigate the effects of ovarian tissue cryopreservation by needle immersed vitrification (NIV) method and subsequently orthotopic transplantation on ovarian function reconstruction in chemotherapy-induced ovary damage rat model. Methods A total of 52 matured virginal female Wistar rats at age of 8-9 weeks housed in specific-pathogen-free facilities, weighing 250-300 g. Vaginal smears were obtained daily, 50 rats having at least 2 consecutive normal estrous cycles were included in the experiment. Ten rats were selected as donors randomly, and NIV method was used for cryopreserving ovarian tissues. The remaining 40 rats were divided into 3 groups according to different treatments: cyclophosphamide group (C group, n=14), cyclophosphamide/transplantation group (C/T group, n=12), and control group (NS group, n=14). In C group and C/T group, the rats received peritoneal injection of cyclophosphamide every day for 21 days to establish the chemotherapy-induced ovary damage models; and then the frozen-thawed ovarian tissues orthotopically transplanted into the left ovarian bursae in C/ T group. The rats received peritoneal injections of 0.9% saline solution every day for 21 days in NS group. Estrous cycle recovery time, ovary weight, morphology change of ovarian tissues, and follicle count were compared among 3 groups. Results One rat died at 2 days after transplantation in C/T group; the other rats survived to the completion of the experiment. At 4 weeks after the end of injection, no significant difference in body weight was found among 3 groups (P gt; 0.05). The rats of NS group had regular estrous cycle, but cyclic changes in vaginal smears were observed in C group and C/T group during cyclophosphamide treatment. The median estrous cycle recovery was 9 days (95%CI: 7.9-10.1 days) in C group, and was 6 days (95%CI: 4.9-7.1 days) in C/ T group, showing significant difference (χ2=6.571, P=0.010). The ovarian weight showed an obvious downtrend in C group at 4 weeks after cyclophosphamide treatment, and an upward trend was observed in C/T group. The ovarian grafts survived and grew well in C/T group. Primordium follicles and primary follicles in C/T group and NS group were significantly more than those in C group (P lt; 0.05), but no significant difference was found between NS group and C/T group (P gt; 0.05). There was no significant difference in secondary follicles and antral follicles among the 3 groups (P gt; 0.05). Conclusion The method of ovarian tissue cryopreservation by NIV and subsequently orthotopic transplantation can significantly shorten the estrous cycle recovery time in chemotherapy-induced ovary damage rat model. Ovarian grafts grow well, follicle count is similar to normal level. So it has the potential ability of ovarian endocrine and fertility reconstruction after chemotherapy.