Objective To systematically isolate and characterize exosomes derived from the mouse melanoma cell line B16F10, reveal their protein profile through proteomic analysis, and evaluate their potential role in oxidative stress regulation and melanin synthesis. Methods Exosomes were isolated from the culture supernatant of B16F10 cells and characterized in terms of physical properties and identification using nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Label-free quantitative proteomic analysis of exosomal proteins was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by functional annotation using bioinformatics methods. Results High-purity exosomes, with typical cup-shaped morphology and an average size of approximately 114.3 nm, were successfully obtained. Proteomic analysis identified 2 249 proteins. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated significant involvement in key pathways such as the insulin signaling pathway and the apoptosis pathway. Exosomes derived from B16F10 cells were enriched in various antioxidant proteins (e.g., peroxiredoxin 1 and glutathione S-transferase pi 1) and key enzymes for melanin synthesis (e.g., tyrosinase-related protein [TYRP] 1, TYRP2, and tyrosinase). Protein-protein interaction network analysis highlighted TYRP1 and TYRP2 as hub proteins. Conclusion B16F10-derived exosomes are naturally loaded with a potent antioxidant protein network and functional enzyme systems for melanin synthesis. This unique molecular composition suggests a dual synergistic mechanism for their potential therapeutic role in vitiligo: the antioxidant components can remodel the redox microenvironment to favor cell survival, while the melanogenic enzymes can directly supplement the core machinery to rapidly restart pigment synthesis. These findings provide a crucial molecular theoretical basis and a library of candidate molecules for developing novel exosome-based, cell-free therapeutic strategies, such as vitiligo treatment.