This study examines the basis of resistance and sensitivity of normal and transformed cells to histone deacetylase inhibitor (HDACi)-induced cell death, specifically the role of caspases and thioredoxin (Trx). An important attribute of HDACis is that they induce cancer cell death at concentrations to which normal cells are relatively resistant, making them well suited for cancer therapy. The mechanism underlying this selectivity has not been understood. In this study we found that the HDACi suberoylanilide hydroxamic acid (SAHA) and MS-275, a benzamide, cause an accumulation of reactive oxygen species (ROS) and caspase activation in transformed but not normal cells. Inhibition of caspases does not block HDACi-induced cell death. These studies provide a possible mechanism that can explain why normal but not certain transformed cells are resistant to HDACi-induced cell death. The HDACi causes an increase in the level of Trx, a major reducing protein for many targets, in normal cells but not in transformed cells. The SAHA-induced increase in Trx activity in normal cells is associated with no increase in ROS accumulation. Transfection of transformed cells with Trx small interfering RNA caused a marked decrease in the level of Trx protein with an increase in ROS, a decrease in cell proliferation, and an increase in sensitivity to SAHA-induced cell death. Thus, Trx, independent of the caspase apoptotic pathway, is an important determinant of resistance of cells to HDACi-induced cell death.