Depsipeptide FR901228 (FK228) is a new kind of histone deacetylase inhibitors (HDACi) that induces growth arrest and cell death in a variety of tumor cells. Though its effects on oncogene expression and degradation have been documented, the detailed mechanism of FK228-induced cytotoxicity is still undefined. In this study, a differential proteomic analysis was performed to identify proteins associated with FK228-induced cytotoxicity in human lung cancer cells. Two-dimensional gel electrophoresis (2-DE) revealed a distinct protein profile of H322 cells in response to FK228 treatment, and 45 protein spots with significant alteration were screened. In total, 27 proteins were identified by mass spectrometry and involved in signal transduction, transcriptional regulation, metabolism, cytoskeletal organization, and protein folding, synthesis and degradation, consistent with multiple effects of FK228 on tumor cells. Notably, a novel target protein, thioredoxin reductase (TrxR), was identified, which is downregulated in FK228-sensitive cancer cells, but upregulated in resistant cells. The expression level of TrxR was negatively correlated with ROS accumulation, DNA damage and apoptosis, implicating TrxR in FK228-induced apoptosis and HDACi sensitivity in cancer cells. Thus, proteomic analysis provides new information about target proteins important for FK228-induced cytotoxicity in cancer cells.