Collagen-tripeptide (CTP) and galacto-oligosaccharide (GOS), which improve collagen homeostasis and barrier function in the skin, are widely used in the food industry to improve wrinkle-related parameters and skin health. In this study, the photoprotective effect of CTP/GOS mixtures (3:1, 1:1, and 1:3) in ultraviolet (UV) B-irradiated hairless mice was examined. Skin parameter analysis, histological approaches, molecular biology techniques and HPLC analysis were applied to investigate the photoaging protective effect, signaling pathways and changes in the microbiota. Oral administration of CTP/GOS mixtures ameliorated photoaged physical parameters and serum levels of pro-inflammatory cytokines compared to UV-irradiated control group. Administration of the 1:3 mixture showed significant changes in the extracellular matrix-related gene expression compared to other mixture groups. The cecal short-chain fatty acid (SCFA) content showed a significant increase in the CTP/ GOS mixed group with a higher GOS content than the control group. In the 16S rRNA-based analysis of cecal microbiota, the relative abundance ratio of the Akkermansia genus belonging to the Verrucomicrobia phylum was higher in CTP and GOS mixture-administered groups than in the UVirradiated control group. Taken together, CTP/GOS mixtures showed a synergistic effect on photoprotective activity through changes in the gene expression, cytokine levels and intestinal microbiota composition.