Under hypoxic conditions, cells activate a transcriptional response mainly driven by hypoxia-inducible factors (HIFs). HIF-1α stabilization and activity are known to be regulated by thioredoxin 1 (Txn1), but how the thioredoxin system regulates the hypoxic response is unknown. By examining the effects of Txn1 overexpression on HIF-1α function in HeLa, HT-29, MCF-7 and EMT6 cell lines, we found that this oxidoreductase did not stabilize HIF-1α, yet could increase its activity. These effects were dependent on the redox function of Txn1. However, Txn1 deficiency did not affect HIF-1α hypoxic-stabilization and activity, and overexpression of thioredoxin reductase 1 (TR1), the natural Txn1 reductase, had no influence on HIF-1α activity. Moreover, overexpression of Txn1 in TR1 deficient HeLa and EMT6 cells was still able to increase HIF-1α hypoxic activity. These results indicate that Txn1 is not essential for HIF-1α hypoxic stabilization or activity, that its overexpression can increase HIF-1α hypoxic activity, and that this effect is observed regardless of TR1 status. Thus, regulation of HIF-1α by the thioredoxin system depends on the specific levels of this system's major components.