Genetic deletion of the adenosine A1 receptor (A1AR) increased renal injury following ischemia–reperfusion injury suggesting that receptor activation is protective in vivo. Here we tested this hypothesis by expressing the human-A₁AR in A₁AR knockout mice. Renal ischemia–reperfusion was induced in knockout mice 2 days after intrarenal injection of saline or a lentivirus encoding enhanced green fluorescent protein (EGFP) or EGFP-human-A₁AR. We found that the latter procedure induced a robust expression of the reporter protein in the kidneys of knockout mice. Mice with kidney-specific human-A₁AR reconstitution had significantly lower plasma creatinine, tubular necrosis, apoptosis, and tubular inflammation as evidenced by decreased leukocyte infiltration, pro-inflammatory cytokine, and intercellular adhesion molecule-1 expression in the kidney following injury compared to mice injected with saline or the control lentivirus. Additionally, there were marked disruptions of the proximal tubule epithelial filamentous (F)-actin cytoskeleton in both sets of control mice upon renal injury, whereas the reconstituted mice had better preservation of the renal tubule actin cytoskeleton, which co-localized with the human-A₁ARs. Consistent with reduced renal injury, there was a significant increase in heat shock protein-27 expression, also co-localizing with the preserved F-actin cytoskeleton. Our findings suggest that selective expression of cytoprotective A₁ARs in the kidney can attenuate renal injury.