BACKGROUND: Chronic hand eczema is a frequent skin disease with a high socio-economic impact. While some light has been shed on genetic factors predisposing for the disease, little is known on its actual pathogenesis. OBJECTIVES: We therefore set out to systematically and comprehensively analyze the differential protein expression in chronic hand eczema using modern mass spectrometry. METHODS: We performed LC-MS/MS analyses and label-free quantification to analyze the proteomic profile of palmar skin from 12 individuals, 6 hand eczema patients and 6 healthy volunteers. Immunohistochemistry of palmar skin from 7 different hand eczema patients and 7 different healthy volunteers was performed in a second step. RESULTS: With this method we were able to identify 185 candidate proteins whose abundance was significantly different in the hand eczema samples. Among them we found several barrier proteins: filaggrin, filaggrin2 and hornerin all were down-regulated in the hand eczema samples as were the desquamation-related enzymes kallikrein-related peptidase 5 and 7 as well as cystatin E/M. The antimicrobial peptides S100A7 and S100A8/A9 as well as the small proline-rich protein 2B and S100A11 were up-regulated in diseased skin. Immunohistochemistry confirmed these findings. CONCLUSIONS: Our results corroborate the assumption that skin barrier dysfunction plays an essential role in the pathogenesis of chronic hand eczema.