Malignant mesothelioma (MM) is a rare tumor associated with occupational exposure to asbestos with no effective treatment regime. Evaluation of mitochondrial function in human MM cell lines revealed a common tumor phenotype: in comparison to immortalized or primary human mesothelial cells, MM tumor cells displayed a more oxidized mitochondrial environment, increased expression of mitochondrial antioxidant enzymes, and altered mitochondrial metabolism. Earlier work by our laboratory indicated that increases in mitochondrial reactive oxygen species (mROS) in MM cell lines supports expression of FOXM1, an oncogenic transcription factor that contributes to increased cell proliferation and chemoresistance. These studies sought to investigate targeting of mitochondrial structure and function as a therapeutic avenue in MM. MM cells have reduced mitochondrial reserve capacity, a redox vulnerability exploitable by pro-oxidant therapeutics. Targeting of the mitochondrial peroxidase peroxiredoxin 3 (PRX3) with the anti-cancer compound thiostrepton (TS) induces irreversible modifications to PRX3 protein, increased mROS, and selective MM cell death. Mass spectrometry showed TS targets conserved cysteine residues in PRX3. In vitro and in MM cells, TS failed to modify human PRX3 harboring mutations to Cys108, Cys127 or Cys229. Pre-incubation of MM cells with dimedone blocked cysteine adduction of PRX3 by TS, suggesting adduction requires an active PRX3 catalytic cycle. Studies with immortalized and primary human mesothelial cells showed adduction of PRX3 by TS occurred at a much lower rate in normal cells than MM cells, and this difference correlated with markedly decreased cytotoxicity. Moreover, MM cells transduced with shRNA to PRX3 grew more slowly and were less sensitive to TS than their wild type counterparts, indicating PRX3 is a major target of TS in MM cells. Studies with a xenoplant mouse model of MM showed TS alone or in combination with the TRX2 inhibitor gentian violet significantly reduced tumor volume. Tumor cell mitochondria have an increased mitochondrial membrane potential, therefore numerous drugs have been developed that selectively accumulatte into energized mitochondria to enhance drug efficacy and specificity. Here two mitochondrial-targeted nitroxides, Mito-carboxy-proxyl (MCP) and Mito-TEMPOL (MT), were investigated for their anti-cancer effects. Treatment of MM cells with MCP or MT led to rapid disruption of the mitochondrial reticulum, increased oxidant levels, and reduced FOXM1 and PRX3 protein expression. Immunostaining revealed a pool of cytoplasmic FOXM1 associated with PRX3 in mitochondria, suggesting PRX3 participates in regulating FOXM1 expression. Combination of MCP or MT with TS led to synergistic effects on MM cell viability. Upregulation of mitochondrial antioxidant enzymes is an adaptive response that ameliorates mitochondrial oxidative stress and supports tumor cell survival. Studies here indicate that enhanced dependency on the PRX3 catalytic cycle in tumor cells promotes inactivation of PRX3 by TS, providing a therapeutic window dependent on a mitochondrial phenotype common to many human tumor types. Therefore TS, alone or in combination with other agents, may prove useful in the management of intractable tumors such as MM.