Mycosporine-glycine (M-Gly), a member of the mycosporine-like amino acid (MAA) family, is known for its potent antioxidant and anti-inflammatory properties. However, its in vivo efficacy in alleviating acute skin photodamage, primarily caused by oxidative stress, has not been well explored. In this investigation, 30 female ICR mice were divided into four groups: a control group and three Ultraviolet B (UVB)-exposed groups treated with saline or M-Gly via intraperitoneal injection for 30 days. At the end of the experiment, UVB exposure caused erythema, wrinkling, collagen degradation, and mast cell infiltration in mouse dorsal skin. M-Gly treatment improved skin appearance and reduced mast cell numbers, while also elevating antioxidant levels, including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). Furthermore, M-Gly reduced inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1β, typically upregulated after UVB exposure. M-Gly also protected skin collagen by upregulating type I procollagen and decreasing MMP-1 levels. Skin metabolomic profiling identified 34 differentially abundant metabolites, and transcriptomic analysis revealed 752 differentially expressed genes. The combined metabolomic and transcriptomic data indicate that M-Gly's protective effects may involve the regulation of ion transport, cellular repair, metabolic stability, collagen preservation, and the Nrf2/HO-1 pathway. These findings highlight M-Gly's potential as an endogenous antioxidant for protecting skin from UVB-induced damage.