Hydrogen peroxide acts as a signaling molecule in early adipogenesis. In differentiating adipocytes, elevated hydrogen peroxide generation is balanced through induction of antioxidant enzymes such as catalase and peroxiredoxins. Thioredoxin reductases (TrxR) and glutathione peroxidases (GPx) are selenoenzymes that constitute part of the major thiol-dependent antioxidant systems in cells. Here we show that the protein levels of cytoplasmic/nuclear TrxR1 and mitochondrial TrxR2 increase in the course of adipocyte differentiation of 3T3-L1 cells together with the TrxR2 substrate thioredoxin 2 (Trx2), resulting in elevated TrxR activity in mature adipocytes. Gene and protein expression of the GPx isoenzyme GPx4 was also stimulated during adipogenesis. Chronic exposure of 3T3-L1 cells to the anti-adipogenic factors tumor necrosis factor α (TNF-α) or rapamycin during differentiation suppressed TrxR1 and Trx2 upregulation, concomitantly with inhibition of adipogenesis and lipogenesis. In contrast, TNF-α or rapamycin did not affect expression of TrxRs and their Trx substrates in mature adipocytes. These results indicate that upregulation of the thioredoxin-dependent redox system is linked to the development of an adipocyte phenotype.