OBJECTIVE - Cellular redox balance is regulated by enzymatic and nonenzymatic systems and freely diffusible nitric oxide (NO) promotes antioxidative mechanisms. We show the NO-dependent transcriptional regulation of the antioxidative thioredoxin system. METHODS AND RESULTS - Incubation of rat pulmonary artery smooth muscle cells (RPaSMC) with the NO donor compound S-nitroso-glutathione (GSNO, 100 μmol/L) suppressed thioredoxin-interacting protein (Txnip), an inhibitor of thioredoxin function, by 71±18% and enhanced thioredoxin reductase 2.7±0.2 fold (n=6; both P<0.001 versus control). GSNO increased thioredoxin activity (1.9±0.5-fold after 4 hours; P<0.05 versus control). Promoter deletion analysis revealed that NO suppression of Txnip transcription is mediated by cis-regulatory elements between -1777 and -1127 bp upstream of the start codon. Hyperglycemia induced Txnip promoter activity (3.9±0.2-fold; P<0.001) and abolished NO effects (-37.4±1.0% at 5.6 mmol/L glucose versus 12.4±2.1% at 22.4 mmol/L glucose; P<0.05). Immunoprecipitation experiments demonstrated that GSNO stimulation and mutation of thioredoxin at Cys69, a site of nitrosylation, had no effect on the Txnip/thioredoxin interaction. CONCLUSIONS - NO can regulate cellular redox state by changing expression of Txnip and thioredoxin reductase. This represents a novel antioxidative mechanism of NO independent of posttranslational protein S-nitrosylation of thioredoxin. © 2006 American Heart Association, Inc.