Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age, whereas the pathogenesis is still not fully understood. Systemic and ovarian oxidative stress (OS) imbalance is a pivotal feature of PCOS. Humanin, a mitochondria derived peptide, has been reported to function as an antioxidant in cardiomyocytes, pancreatic beta cells, and other cells, but how this function is regulated remains unclear. In this study, we investigated whether humanin expression differs in the granulosa cells of PCOS patients versus controls, and whether humanin alleviates oxidative stress in PCOS ovaries. Sixteen PCOS patients and twenty-eight age and BMI-matched controls undergoing IVF were recruited, and their serum, follicular fluid and granulosa cells were collected for humanin analysis. Dehydroepiandrosterone-induced rat PCOS models, and vitamin K3 (vitK3)-induced OS COV434 cell lines were to investigate the mechanism. Humanin expression was significantly down-regulated in the ovaries of PCOS patients relative to those of non-PCOS patients. Exogenous humanin supplementation significantly attenuated body weight gain, ovarian morphological abnormalities, endocrinological disorders and ovarian and systemic OS in PCOS rat models. Our study further demonstrated that this attenuation effect was involved in the modulation of the Keap1/Nrf2 signalling pathway. In summary, this study reported for the first time that decreased expression of humanin in the granulosa cells was associated with oxidative imbalance in PCOS. Humanin alleviates oxidative stress in ovarian granulosa cells of PCOS patients via modulation of the Keap1/Nrf2 signaling pathway. © The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.