Hepatic insulin resistance is an important pathophysiology in type 2 diabetes, and the mechanisms by which high-caloric diets induce insulin resistance are unclear. Among vertebrate animals, mammals have retained a unique molecular change that allows an intracellular arrestin domain-containing protein called Thioredoxin-Interacting Protein (TXNIP) to bind covalently to thioredoxin, allowing TXNIP to "sense" oxidative stress. Here, we show that a single cysteine in TXNIP mediates the development of hepatic insulin resistance in the setting of a high-fat diet (HFD). Mice with an exchange of TXNIP Cysteine 247 for Serine (C247S) showed improved whole-body and hepatic insulin sensitivity compared with WT controls following an 8-week HFD. HFD-fed TXNIP C247S mouse livers also showed improved insulin signaling. The Transmembrane 7 Superfamily Member 2 (Tm7sf2) gene encodes for a sterol reductase involved in the process of cholesterol biosynthesis. We identified TM7SF2 as a potential mediator of enhanced insulin signaling in HFD-fed TXNIP C247S mouse livers. TM7SF2 increased Akt phosphorylation and suppressed gluconeogenic markers PCK1 and G6Pc specifically under oxidative stress-induced conditions in HepG2 cells. We also present data suggesting that a heterozygous variant of TXNIP C247 is well tolerated in humans. Thus, mammals have a single redox-sensitive amino acid in TXNIP that mediates insulin resistance in the setting of an HFD. Our results reveal an evolutionarily conserved mechanism for hepatic insulin resistance in obesity. Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.