Objective The comparative proteomic study on the paired lesional epidermis (LE) and non-lesional epidermis (NLE) from vitiligo patients to identify the differentially expressed proteins (DEPs) between LE and NLE, and to further explore the molecular mechanism of pathogenesis of vitiligo.Methods Firstly, the in solution digestion condition for proteins from epidermis were optimized to sequential tandem digestion with Lys-C and trypsin. Secondly, tandem mass tag (TMT) based quantitative proteomic strategy was performed to compare the proteome profile of the paired LE and NLE from three stable non-segmental vitiligo subjects, and differential expressed proteins (DEPs) were identified. At last, the functional enrichment analysis was performed via bioinformatics tool and database (GO, KEGG, STRING, GSEA).Results The optimal sequential tandem digestion condition was the combination of Lys-C (enzyme∶substrate, 1∶100) and trypsin (enzyme∶substrate, 1∶50). A total of 4 496 proteins were identified, and of which 181 were DEPs between LE and NLE from vitiligo patients. Bioinformatics analysis showed that DEPs were mainly related with metabolism, immunity, redox and cell adhesion. Among them, the 119 up-regulated proteins are mainly involved in the processes of keratinization, transcription, oxidative stress, and proteolysis. The 62 down-regulated proteins are mainly involved in intracellular transport, glutathione metabolism and actin filament capping.Conclusion The comparative proteomic study revealed that there were functional differences in keratinization, immunity, lipid metabolism and redox between LE and NLE in vitiligo patients. PRDX1, PRDX2, EEF2, ITGB1, SPTBN2, ANXA1 and PFKL were found as the key proteins to disfunction of LE.