Skin is an important component of the mucosal immune system in fish, however its molecular mechanism at the proteome-level remains unclear. In this study, differentially expressed proteins in the skin of zebrafish Danio rerio responding to Aeromonas hydrophila infection were identified using two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) analysis. The results showed that 17 proteins were differentially expressed, six proteins of which were up-regulated and 11 proteins down-regulated. MHC class II DR α 1 domain, immunoglobulin heavy chain V-region, glyceraldehyde-3-phosphate dehydrogenase, transcription factor AP-2-α, and actin were considered immune-related molecules in the skin of zebrafish. Expression changes of 17 proteins were further determined by zebrafish cDNA microarray analysis. This is the first known report on proteome of fish skin against A. hydrophila, which will contribute to the understanding of the mucosal immune response, bacterial disease control and prevention.