Renal ischemia-reperfusion injury is a major cause of acute kidney injury. We previously found that renal A(1) adenosine receptor (A(1)AR) activation attenuated multiple cell death pathways including necrosis, apoptosis, and inflammation. Here, we tested whether induction of cytoprotective sphingosine kinase (SK)-1 and sphingosine-1-phosphate (S1P) synthesis might be the mechanism of protection. A selective A(1)AR agonist (CCPA) increased the synthesis of S1P and selectively induced SK1 in mouse kidney and HK-2 cells. This agonist failed to protect SK1-knockout but protected SK2-knockout mice against renal ischemia-reperfusion injury indicating a critical role of SK1 in A(1)AR-mediated renal protection. Inhibition of SK prevented A(1)AR-mediated defense against necrosis and apoptosis in HK-2 cells. A selective S1P(1)R antagonist (W146) and global in vivo gene knockdown of S1P(1)Rs with small interfering RNA completely abolished the renal protection provided by CCPA. Mice selectively deficient in renal proximal tubule S1P(1)Rs (S1P(1)R(f)(/)(f) PEPCK(Cre/-)) were not protected against renal ischemia-reperfusion injury by CCPA. Mechanistically, CCPA increased nuclear translocation of hypoxia-inducible factor-1α in HK-2 cells and selective hypoxia-inducible factor-1α inhibition blocked A(1)AR-mediated induction of SK1. Thus, proximal tubule SK1 has a critical role in A(1)AR-mediated protection against renal ischemia-reperfusion injury.