Aims: Methionine sulfoxide reductase A (MsrA) and methionine metabolism are associated with oxidative stress, a principal cause of ischemia/reperfusion (I/R) injury. Herein, we investigated the protective role of MsrA against kidney I/R injury and the involvement of MsrA in methionine metabolism and the trans-sulfuration pathway during I/R. Results: We found that MsrA gene-deleted mice (MsrA^−/−) were more susceptible to kidney I/R injury than wild-type mice (MsrA^+/+). Deletion of MsrA enhanced renal functional and morphological impairments, congestion, inflammatory responses, and oxidative stress under I/R conditions. Concentrations of homocysteine and H₂S in the plasma of control MsrA^−/− mice were significantly lower than those in control MsrA^+/+ mice. I/R reduced the levels of homocysteine and H₂S in both MsrA^+/+ and MsrA^−/− mice, and these reductions were significantly more profound in MsrA^−/− than in MsrA^+/+ mice. I/R reduced the expression and activities of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), both of which are H₂S-producing enzymes, in the kidneys. These reductions were more profound in the MsrA^−/− mice than in the MsrA^+/+mice. Innovation: The data provided herein constitute the first in vivo evidence for the involvement of MsrA in regulating methionine metabolism and the trans-sulfuration pathway under normal and I/R conditions. Conclusion: Our data demonstrate that MsrA protects the kidney against I/R injury, and that this protection is associated with reduced oxidative stress and inflammatory responses. The data indicate that MsrA regulates H₂S production during I/R by modulating the expression and activity of the CBS and CSE enzymes. Antioxid. Redox Signal. 18, 2241—2250.