Aims: Mitochondrial thioredoxin reductase (Txnrd2) is a central player in the control of mitochondrial hydrogen peroxide (H₂O₂) abundance by serving as a direct electron donor to the thioredoxin-peroxiredoxin axis. In this study, we investigated the impact of targeted disruption of Txnrd2 on tumor growth. Results: Tumor cells with a Txnrd2 deficiency failed to activate hypoxia-inducible factor-1α (Hif-1α) signaling; it rather caused PHD2 accumulation, Hif-1α degradation and decreased vascular endothelial growth factor (VEGF) levels, ultimately leading to reduced tumor growth and tumor vascularization. Increased c-Jun NH2-terminal Kinase (JNK) activation proved to be the molecular link between the loss of Txnrd2, an altered mitochondrial redox balance with compensatory upregulation of glutaredoxin-2, and elevated PHD2 expression. Innovation: Our data provide compelling evidence for a yet-unrecognized mitochondrial Txnrd-driven, regulatory mechanism that ultimately prevents cellular Hif-1α accumulation. In addition, simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems was used as an efficient therapeutic approach in hindering tumor growth. Conclusion: This work demonstrates an unexpected regulatory link between mitochondrial Txnrd and the JNK-PHD2-Hif-1α axis, which highlights how the loss of Txnrd2 and the resulting altered mitochondrial redox balance impairs tumor growth as well as tumor-related angiogenesis. Furthermore, it opens a new avenue for a therapeutic approach to hinder tumor growth by the simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems. Antioxid. Redox Signal. 22, 938–950.