The role of A₃ adenosine receptors (ARs) in sepsis and inflammation is controversial. In this study, we determined the effects of A₃AR modulation on mortality and hepatic and renal dysfunction in a murine model of sepsis. To induce sepsis, congenic A₃AR knockout mice (A₃AR KO) and wild-type control (A₃AR WT) mice were subjected to cecal ligation and double puncture (CLP). A₃AR KO mice had significantly worse 7-day survival compared with A₃AR WT mice. A₃AR KO mice also demonstrated significantly higher elevations in plasma creatinine, alanine aminotransferase, aspartate aminotransferase, keratinocyte-derived chemokine, and TNF-α 24 h after induction of sepsis compared with A₃AR WT mice. Renal cortices from septic A₃AR KO mice exhibited increased mRNA encoding proinflammatory cytokines and enhanced nuclear translocation of NF-kB compared with samples from A₃AR WT mice. A₃AR WT mice treated with N⁶-(3-iodobenzyl)ADO-5′N-methyluronamide (IB-MECA; a selective A₃AR agonist) or 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191; a selective A₃AR antagonist) had improved or worsened 7-day survival after induction of sepsis, respectively. Moreover, A₃AR WT mice treated with IB-MECA or MRS-1191 showed acutely improved or worsened, respectively, renal and hepatic function following CLP. IB-MECA significantly reduced mortality in mice lacking the A₁AR or A_2aAR but not the A₃AR, demonstrating specificity of IB-MECA in activating A₃ARs and mediating protection against sepsis-induced mortality. We conclude that endogenous or exogenous A₃AR activation confers significant protection from murine septic peritonitis primarily by attenuating the hyperacute inflammatory response in sepsis.