Ultraviolet B (UVB)-induced cell death causes skin photoaging. In this study, we investigated the protective effect of Melanogrammus aeglefinus skin oligopeptide (MSOP) in UVB-irradiated human keratinocytes. The method of preparing MSOP was optimized, and three peptides with high abundance, VADML (Val-Ala-Asp-Met-Leu), IARF (Ile-Ala-Arg-Phe) and SSPSF (Ser-Ser-Pro-Ser-Phe), were identified. Discovery Studio predicted that these peptides interacted with Keap1 and contributed to antioxidant activity. Therefore, a UVB-induced cell model was used to explore the beneficial effects of MSOP in vitro. The activities of superoxide dismutase and glutathione peroxidase were increased in the MSOP-treated groups, while the malondialdehyde content was decreased. In addition, 23 differentially expressed proteins were identified through quantitative proteomics analysis; among them, the upregulation of Nrf2 and downregulation of Keap1, which are involved in the Keap1/Nrf2/ARE signaling pathway, contributed to the antioxidant process. Based on this study, MSOP might be an alternative agent for protecting the skin against UVB exposure.