The role of monocytes/macrophages in the pathogenesis of ischemia-reperfusion injury (IRI) is unknown. We sought to determine whether activation of macrophage adenosine 2A (A_2A) receptors (A_2ARs) mediates tissue protection. We subjected C57Bl/6 mice infused with clodronate [dichloromethylene bisphosphonate (Cl₂MBP)] to IRI (32 min of ischemia followed by 24 h of reperfusion) to deplete them of macrophages. IRI induced an elevation of plasma creatinine that was reduced with Cl₂MBP (26% of control). Adoptive transfer of murine RAW 264.7 cells reconstituted injury, an effect blocked significantly by A_2A agonists (27% of plasma creatinine from mice reconstituted with macrophages). Macrophages subjected to A_2A knockout by small interfering RNA were adoptively transferred to macrophage-depleted mice and reconstituted injury (110% of control mice); however, the increase in plasma creatinine was blocked by A_2A agonists (20% of vehicle treatment). Finally, the A_2A agonist effect on IRI was blocked in macrophage-depleted A_2A-knockout mice reconstituted with wild-type RAW 264.7 cells. RNase protection assays 24 h after IRI demonstrated that macrophages are required for IL-6 and TGF-β mRNA induction. However, A_2A agonist-mediated tissue protection is independent of IL-6 and TGF-β mRNA. We conclude that the full extent of IRI requires macrophages and that A_2A agonist-mediated tissue protection is independent of activation of macrophage A_2ARs.