Ovarian tissue cryopreservation and transplantation (OCT) has been sufficiently proven effective and feasible to preserve fertility for women especially for prepubertal girls suffering from cancer with radiotherapy and chemotherapy. However, grafts' survival, significant follicle loss and a delay of revascularization during OCT still need to be resolved no matter what kind of cryopreserved method being used. Different from previous reports about additives treatment on recipient after ovarian transplantation, we here report a new vitrification protocol with pretreatment of rapamycin, an inhibitor of the mTOR signaling pathway. The rapamycin treatment has been shown to inhibit the activation of mTOR signaling pathway in fresh thawed ovaries or in ovaries shortly grafted in the recipient mice. Further study revealed increased percentage of primordial follicles and reduced apoptosis after 5 days of transplantation. Long-term follow up of ovarian development demonstrated the increase of ovarian survival rates in rapamycin treated ovaries after 2 weeks of transplantation. Although follicular development showed a slight delay with more secondary and early antral follicles found in rapamycin treated ovaries, follicular development was not blocked as manifested by the ovarian morphology after 5 weeks of transplantation. Taken together, the pretreatment of rapamycin before vitrification is a good method for clinical application with its effectiveness on preserving follicle reserve and promoting ovarian survival during the process of OCT. (C) 2020 Published by Elsevier Inc.