OBJECTIVE This study was performed to investigate the effect of hypothermic machine perfusion (HMP) after cold storage (CS) on ovarian transplantation. METHODS Rats aged 8-10 weeks were used as the donors and recipients for allotransplantation. Eighteen donor rats were divided into three groups: the fresh control (n = 6), cold storage (CS; n = 6), and hypothermic machine perfusion (HMP; n = 6) groups. The preservation solution contained Dulbecco's modified Eagle's medium/Ham's F-12 (1:1, v/v), 10% fetal bovine serum, 10 μg/ml insulin, 10 μg/ml transferrin, and 50 mIU/ml follicle-stimulating hormone (FSH). The donor ovaries in the CS and HMP groups were excised and then respectively subjected to 4 h of CS and 2 h of CS combined with 2 h of HMP at 4 °C, and then transplanted beneath the recipient's left renal capsule. At 7 days after transplantation, the ovaries were removed and blood samples were obtained for histological analysis, immunohistochemistry for CD31 and Ki67, and serum anti-Mullerian hormone (AMH) level estimation. RESULTS The HMP group showed significant increases in serum AMH and CD31-positive areas when compared to these values in the CS group (P < 0.05). However, no differences were noted in the total number of follicles or the Ki67-positive areas among the three groups. CONCLUSIONS Hypothermic machine perfusion after static cold storage is more effective than static CS alone for the short-term preservation of whole ovaries during transport. Whole ovary transplantation with vascular pedicle is our future research direction. Graphical Abstract The black rectangle in the figure shows the place where ligation and disconnection are required, the black dotted line shows the place where vascular forceps are used to clamp, and the black circle shows the place where the cannula is inserted This diagram was made for reviewers to understand more intuitively how my hypothermia mechanical perfusion model was built. Organs obtained in this way can be used for subsequent perfusion and whole ovarian transplantation.