PURPOSE: The Nrf2/Keap1/ARE pathway is a major regulator of cytoprotective responses to oxidants. Gluatredoxin-1 (Glrx-1), a small thiol transferase removes glutathione (GSH) adducts from proteins and participates in redox signaling. Glrx-/- mice exhibit increased protein GSH adducts (PSSG) and non-alcoholic fatty liver disease (NAFLD). Unexpectedly, our Glrx-/- mice showed increased hepatic glutathione (GSH) levels. The Nrf2/Keap1/ARE pathway, as an important regulator of glutathione synthesis, could be regulated by Glrx-1 activity. METHODS: To determine the role of Nrf2 in vivo, we treated Glrx-/- mice with high fat high sucrose (HFHS) diet to induce metabolic and oxidative stress. Livers were harvested at 10 months of age after 8 months on HFHS diet. Gene expression of Nrf2 and its down-signaling targets were determined using RT-qPCR and protein expression was accessed via WB. To determine the role of Nrf2 in Glrx-deficiency in vitro, Glrx siRNA was transfected in HEK293A and HepG2 cells and exposed to high palmitate high glucose (HPHG) to mimic metabolic stress and hydrogen peroxide to mimic oxidative stress. RESULTS: Glrx-/- deficiency increased Nrf2 activity and gene expression, and decreased Keap1 activity and gene expression. Glrx silencing in liver promoted Nrf2 activity and translocation to the nucleus, and downstream targets of Nrf2 were upregulated. CONCLUSION: Our findings indicate that the Nrf2/Keap1/ARE pathway is regulated by Glrx in vitro and in vivo.