Objective: Human kidney proximal tubule epithelial HK-2 cells were treated with either 2-ME (10 to 1000 µM) or vehicle at 60 minutes before exposure to 0.3 mM H2O2 for 1 to 6 hours. Cell viability was measured by MTT assay. Bilateral IRI or sham-operation in male C57BL/6 mice was carried out. Twenty-four hours before IRI or sham-operation, mice were intraperitoneally and daily treated with 2-ME (3 to 30 mg/kg body weight per day) or vehicle. Plasma creatinine concentration was measured as represented by kidney function. Phospho-S139 H2A histone family member X (γH2AX) expression, ataxia telangiectasia mutated (ATM) phosphorylation, and ATM- and Rad3-related (ATR) phosphorylation were measured by western blot analysis.   Methods: Treatment with 2-ME dose- and time-dependently decreased cell death induced by H2O2 injury in HK-2 cells. IRI-induced loss of renal function was improved by treatment with 2-ME as demonstrated by reduced plasma creatinine level. In addition, treatment with 2-ME significantly attenuated histological injury after IRI in kidney tubules. IRI remarkedly upregulated γH2AX, a DNA damage marker, but treatment with 2-ME significantly reduced the upregulation of γH2AX. Furthermore, phosphorylated ATM and ATR, DNA damage response sensor kinases, were increased by treatment with 2-ME after IRI. During IRI, treatment with 2-ME significantly more increased.   Results: These data suggest that 2-ME protects against IRI-induced DNA damage injury through enhancing DNA damage response sensor kinases including ATM and ATR.   Conclusions: Objective: Kidney ischemia and reperfusion injury (IRI) is a major cause of acute kidney injury (AKI), resulting in a high mortality rate. DNA damage is occurs in various conditions including irradiation, oxidative stress, and IRI. 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. Here, we tested whether 2-ME prevents kidneys IRI.   Methods: Human kidney proximal tubule epithelial HK-2 cells were treated with either 2-ME (10 to 1000 µM) or vehicle at 60 minutes before exposure to 0.3 mM H2O2 for 1 to 6 hours. Cell viability was measured by MTT assay. Bilateral IRI or sham-operation in male C57BL/6 mice was carried out. Twenty-four hours before IRI or sham-operation, mice were intraperitoneally and daily treated with 2-ME (3 to 30 mg/kg body weight per day) or vehicle. Plasma creatinine concentration was measured as represented by kidney function. Phospho-S139 H2A histone family member X (γH2AX) expression, ataxia telangiectasia mutated (ATM) phosphorylation, and ATM- and Rad3-related (ATR) phosphorylation were measured by western blot analysis.   Results: Treatment with 2-ME dose- and time-dependently decreased cell death induced by H2O2 injury in HK-2 cells. IRI-induced loss of renal function was improved by treatment with 2-ME as demonstrated by reduced plasma creatinine level. In addition, treatment with 2-ME significantly attenuated histological injury after IRI in kidney tubules. IRI remarkedly upregulated γH2AX, a DNA damage marker, but treatment with 2-ME significantly reduced the upregulation of γH2AX. Furthermore, phosphorylated ATM and ATR, DNA damage response sensor kinases, were increased by treatment with 2-ME after IRI. During IRI, treatment with 2-ME significantly more increased.   Conclusions: These data suggest that 2-ME protects against IRI-induced DNA damage injury through enhancing DNA damage response sensor kinases including ATM and ATR.