Photo-aging is caused by cumulative oxidative stress from ultraviolet B irradiation with up-regulating intracellular reactive oxygen species, 4-hydroxynonenal, and matrix metalloproteinases. MMPs are the enzyme that degrades collagens so that impair the function of the dermis. Galangin was identified by 1H-NMR and 13C-NMR spectroscopy and is a natural flavonol that recently known to have many pharmacological effects such as anti-viral, anti-inflammatory, anti-atopic dermatitis and anti-oxidative activities. In this study, the protective effect of galangin on UVB-induced photo-aging in human skin fibroblasts (CCD-986sk) was conducted by Western blot analysis and enzyme-linked immunosorbent assay. Activator protein 1 and nuclear factor-kappa B are the main transcription factors from activated mitogen-activated protein kinases that up-regulates MMPs. Galangin showed down-regulation of intracellular ROS, 4-HNE, and MMPs through inhibition of phosphorylation of the MAPK pathway and showed a protective effect against skin fibroblasts under oxidative stress caused by UVB irradiation. This lead to up-regulation of fibroblast growth factor 2 and type 1 pro-collagen. These findings suggest that galangin can be developed as a potential agent for functional food and cosmetics of UVB-induced skin photo-aging.