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Skin provides the protective barrier for our body and undergoes the continuous regeneration in order to overcome damage from exposure to harmful environments and wounds. Epidermal stem cells (ESCs) play critical roles in skin regeneration. Humanin analogue, S14G-humanin (HNG), a prominent member of a newly discovered family of mitochondrial-derived peptides, has been shown to be a cytoprotective derivative in multiple cell types. In this study, we isolated mouse epidermal stem cells and investigated the cytoprotective effects of HNG on ESCs upon ultraviolet (UV)-B treatment. We show that HNG suppresses UV-B-induced ROS production and increases antioxidant glutathione expression. HNG-pretreated cells exhibit very mild production of cytokines, including TNF-α, IL-1β, and IL-6, upon exposure to UV-B. HNG pretreatment is protective against UV-B-mediated cytotoxicity and promotes ESC survival. Moreover, HNG treatment attenuates the UV-B-induced reduction in mitochondrial membrane potential (MMP) and preserves their identity and stem cell capacity. Mechanistically, HNG treatment ameliorates the UV-B-induced reduction in Wnt/β-catenin pathway proteins, including Wtn3a, Myc, and cyclin D1. Collectively, our data suggest that HNG acts as a pro-survival and anti-oxidative stress agent in ESCs and has the potential to be used in ESC-mediated therapies., (Copyright © 2018. Published by Elsevier Masson SAS.)