Hypoxia is a key driver of tumor angiogenesis. While the roles of HIF1a and prolylhydroxylases (PHDs) in oxygen sensing and tumor angiogenesis have been well established, little is known regarding a possible involvement of mitochondrial reactive oxygen species (ROS) in tumor progression. The mitochondrial thioredoxin system consists of thioredoxin, thioredoxin reductase (Txnrd2) and peroxiredoxins, and is one of the primary antioxidant defence systems controlling mitochondrial ROS. Since ROS are considered to be implicated in multiple steps during tumorigenesis, we examined whether loss of Txnrd2 may affect tumor growth and tumor angiogenesis. Here we show that in vitro cultured c-myc and ha-ras transformed Txnrd2 null fibroblasts have a strongly impaired clonogenic potential compared to wild-type control cells. When transplanted subcutaneously into mice, tumor growth was reduced by as much as 50%. Txnrd2 knockout tumors displayed a delayed angiogenic switch, a strongly diminished tumor vascularization, and impaired tumor perfusion. Knockout tumors revealed decreased HIF1a and vascular endothelial growth factor (VEGF) levels. Transformed Txnrd2 knockout cells failed to stabilise HIF1a in response to starvation in vitro, pointing to an important role of Txnrd2 in HIF1a regulation. When tumor-bearing mice were deprived from intracellular glutathione by pharmacological inhibition of glutathione synthesis, Txnrd2 knockout tumors showed a further reduction in tumor growth. In summary, these studies reveal that mitochondrial ROS metabolism has a major influence in tumor growth and tumor angiogenesis, providing a potential therapeutic rationale to hinder tumor growth.