Premature ovarian insufficiency (POI) is a major cause of female infertility, for which effective therapies remain limited. S14G-Humanin (HNG), a potent analogue of Humanin, exhibits strong antioxidant and anti-apoptotic properties, and has demonstrated cytoprotective effects in various tissues, including the ovary. In this study, a cyclophosphamide-induced POI (CP-POI) mouse model was established to evaluate both the ovarian damage induced by chemotherapy and the protective effects of HNG. HNG administration significantly increased the numbers of primordial follicles (P = 0.044) and growing follicles (all P > 0.05), as well as corpora luteum (P = 0.09). Moreover, HNG markedly improved oocyte quality (P = 0.009), significantly lowering the proportion of abnormal ovulated oocytes (P = 0.002). Fertility outcomes were also enhanced: CP treatment significantly reduced litter size compared to controls (4.6 ± 1.1 vs. 8.0 ± 1.0; P < 0.001), whereas HNG treatment significantly mitigated this reduction (6.2 ± 0.8 vs. 4.6 ± 1.1; P = 0.029). Mechanistically, HNG alleviated oxidative stress and apoptosis in ovarian tissues (all P < 0.05), reduced ROS levels (P = 0.034), and restored mitochondrial membrane potential (P = 0.004) in a human granulosa cell line. Furthermore, HNG significantly upregulated PGC-1α expression and enhanced AMPK phosphorylation in both in vivo and in vitro models (both P < 0.05). Collectively, these findings demonstrate that HNG confers significant protection against chemotherapy-induced ovarian damage and highlight its potential as a novel therapeutic agent for chemotherapy-induced ovarian damage.© The Author(s) 2025. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.