Thioredoxin 1 (Trx) is a known redox regulator that is implicated in the redox control of cell growth and apoptosis inhibition. Here we show that Trx is essential for maintaining the content of S-nitrosylated molecules in endothelial cells. Trx itself is S-nitrosylated at cysteine 69 under basal conditions, and this S-nitrosylation is required for scavenging reactive oxygen species and for preserving the redox regulatory activity of Trx. S-nitrosylation of Trx also contributes to the anti-apoptotic function of Trx. Thus, Trx can exert its complete redox regulatory and anti-apoptotic functions in endothelial cells only when cysteine 69 is S-nitrosylated.