Objectives: Kidney ischemia and reperfusion injury (IRI) contributes to DNA damage.  2-Mercaptoethanol (2-ME) acts as an antioxidant by scavenging hydroxyl radicals, while it is considered toxic.  However, a role of 2-ME in kidney IRI remains to be defined. Methods: Bilateral IRI or sham-operation in 8 to 10 week-old male C57BL/6 mice was carried out. The mice were treated with 2-ME (3 to 30 mg/kg body weight per day), Ras-selective lethal 3 (RSL3, a potent inhibitor of glutathione peroxidase 4 (GPX4); 10 mg/kg body weight per day), or saline (vehicle).  Kidney function and tubular injury were assessed by plasma creatinine concentration and percentage of injured tubules, respectively.  GPX4 expression and phosphorylations of H2A.X variant histone phosphorylated at serine 139 (γH2AX), ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 related (ATR), checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), and X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) were measured by western blot analysis. Results: Treatment with 2-ME significantly attenuated renal dysfunction, tubular injury, and DNA damage in IRI-subjected kidneys.  Among DNA damage response kinases, ATM phosphorylation was more increased by 2-ME in IRI kidneys.  ATM-related Chk1, Chk2, and XRCC1 phosphorylations were also increased by 2-ME after IRI.  2-ME dramatically upregulated GPX4 in the kidney subjected to IRI.  Pharmacological inhibition of GPX4 reversed renal dysfunction, tubular injury, DNA damage, and ATM phosphorylation induced by IRI in 2-ME-treated mouse kidneys. Conclusions: These data suggest that 2-ME protects against IRI-induced DNA damage through upregulation of GPX4.