The rodent uterus responds to acute estradiol (E2) treatment with a series of well characterized physiological responses. In a recent screen for genes involved in this response, we found that several genes in the thioredoxin (Txn) pathway were rapidly modified after E2 treatment in the mouse uterus. Txn is a 12-kDa protein with multiple roles in the cell, including protection against oxidative stress and apoptosis, regulation of transcription factor activity, and regulation of cellular proliferation. Txn in combination with Txn reductase (Txnrd) and Txn-interacting protein (Txnip) constitute the mammalian Txn pathway. This pathway exists in multiple locations in the cell, including the cytosol and mitochondria. To analyze the levels of Txn, Txnrd, and Txnip in the uterus, we treated ovariectomized adult mice with a time course of E2 and analyzed mRNA levels by real-time PCR. E2 rapidly decreased the expression of Txnip, but increased the levels of cytosolic Txn1 and Txnrd1 as well as mitochondrial Txn2. Using the ER antagonist, ICI 182,780, and mice lacking functional estrogen receptor alpha (ERalpha), we demonstrate that these E2-mediated changes require ERalpha, but not ERbeta. The repression of Txnip by E2 was also demonstrated in vitro in MCF-7 human breast cancer cells. This repression was blocked by treatment with the histone deacetylase inhibitor, trichostatin A, suggesting that repression by E2 may involve regulation of histone acetylation. We conclude that the rapid E2-mediated activation of the Txn pathway is an important step in the response of the mammalian uterus to estrogen.