Reactive oxygen species are important cellular signaling molecules, and thioredoxin (TRX) is a key regulator of cellular redox balance. We investigated the interaction of TRX with its endogenous inhibitor, vitamin D₃–upregulated protein (VDUP)-1, in human aortic smooth muscle cells (SMCs). Adenoviral gene transfer of TRX enhanced TRX enzyme activity 2.7±0.4-fold (P<0.05 versus cells infected with adenoviral vector expressing green fluorescent protein [AdGFP]) and resulted in a 3.8±0.5-fold increase of cellular DNA synthesis as detected by methyl-[³H]thymidine incorporation (P<0.001). Platelet-derived growth factor (PDGF) also increased TRX enzyme activity 2.5±3.3-fold (P<0.05 versus no stimulation) and DNA synthesis 6.5±0.3-fold (P<0.001 versus no stimulation) without significant changes in TRX expression. PDGF and H₂O₂ time-dependently suppressed VDUP-1 expression (13-fold and 30-fold reduction after 1 hour, respectively; P<0.001), and this was inhibited by the cell-permeable antioxidants N-acetylcysteine and 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron). Overexpression of VDUP-1 (AdVDUP-1) reduced TRX activity at baseline (−61±23% versus control cells, P<0.05) and abolished PDGF-induced TRX activity (−9±27% in AdVDUP-1–infected cells; P=NS versus control cells). In addition, overexpression of VDUP-1 blocked PDGF-induced DNA synthesis (1.3±0.4-fold increase in AdVDUP-1–infected cells versus 6.5±0.4-fold increase in AdGFP-infected cells, P<0.001). In conclusion, VDUP-1 has marked antiproliferative effects in SMCs through the suppression of TRX activity, suggesting that the regulation of VDUP-1 is a critical molecular switch in the transduction of pro-oxidant mitogenic signals. These data also demonstrate that activation of the reductase TRX plays a pivotal role in the redox-dependent proliferation of SMCs.