Prepubertal chemotherapy induced ovarian damage poses a significant threat to female fertility, particularly following cyclophosphamide (CP) treatment. Humanin (HNG), a small molecule polypeptide encoded by mitochondrial DNA, has a variety of effects, this study aimed to investigate the protective effects of HNG and its combination with conventional Gonadotropin Releasing Hormone Agonist (GnRHa) on ovarian function in a CP-induced damage model. The 21-day-old C57BL/6 J female mice were randomly assigned to six groups: Control, CP model, HNG, HNG+CP, GnRHa+CP, and HNG+GnRHa+CP. Reproductive related parameters were assessed through histopathological examination, follicle counts, serum sex hormone levels, estrous cycle monitoring, and oxidative stress evaluation. Results indicated that CP treatment led to significant reproductive dysfunction especially ovarian dysfunction, evidenced by reduced follicles, hormonal imbalances, prolonged estrous cycles, reduced body weight, and diminished ovarian and uterine weights, alongside pathological alterations. Notably, HNG treatment, both alone and in conjunction with GnRHa, significantly mitigated these adverse effects, however the combination did not provide additional benefits over HNG alone regarding follicles preservation and antioxidant capacity. Transcriptomic analysis revealed significant enrichment in inflammation and immune response pathways following HNG treatment. In conclusion, HNG demonstrates potential as a therapeutic agent to protect against CP-induced ovarian damage, offering insights for future strategies aimed at preserving female fertility during chemotherapy.Copyright © 2025. Published by Elsevier Inc.