DC-mediated NKT cell activation is critical in initiating the immune response following kidney ischemia/reperfusion injury (IRI), which mimics human acute kidney injury (AKI). Adenosine is an important antiinflammatory molecule in tissue inflammation, and adenosine 2A receptor (A_2AR) agonists protect kidneys from IRI through their actions on leukocytes. In this study, we showed that mice with A_2AR-deficient DCs are more susceptible to kidney IRI and are not protected from injury by A_2AR agonists. In addition, administration of DCs treated ex vivo with an A_2AR agonist protected the kidneys of WT mice from IRI by suppressing NKT production of IFN-γ and by regulating DC costimulatory molecules that are important for NKT cell activation. A_2AR agonists had no effect on DC antigen presentation or on Tregs. We conclude that ex vivo A_2AR–induced tolerized DCs suppress NKT cell activation in vivo and provide a unique and potent cell-based strategy to attenuate organ IRI.