Controversy exists regarding the effect of A₁ adenosine receptor (AR) activation in the kidney during ischemia and reperfusion (I/R) injury. We sought to further characterize the role of A₁ ARs in modulating renal function after I/R renal injury using both pharmacological and gene deletion approaches in mice. A₁ AR knockout mice (A₁KO) or their wild-type littermate controls (A₁WT) were subjected to 30 min of renal ischemia. Some A₁WT mice were subjected to 30 min of renal ischemia with or without pretreatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or 2-chrolo-cyclopentyladenosine (CCPA), selective A₁ AR antagonist and agonist, respectively. Plasma creatinine and renal histology were compared 24 h after renal injury. A₁KO mice exhibited significantly higher creatinines and worsened renal histology compared with A₁WT controls following renal I/R injury. A₁WT mice pretreated with the A₁ AR antagonist or agonist demonstrated significantly worsened or improved renal function, respectively, after I/R injury. In addition, A₁WT mice pretreated with DPCPX or CCPA showed significantly increased or reduced markers of renal inflammation, respectively (renal myeloperoxidase activity, renal tubular neutrophil infiltration, ICAM-1, TNF-α, and IL-1β mRNA expression), while demonstrating no differences in indicators of apoptosis. In conclusion, we demonstrate that endogenous or exogenous preischemic activation of A₁ ARs protects against renal I/R injury in vivo via mechanisms leading to decreased necrosis and inflammation.