The transcription factor NF-κB has been found critical to the pathogenesis of renal ischemia-reperfusion injury, which is a major cause of acute kidney injury (AKI). Activation of NF-κB is dependent upon the activation of the specific inhibitory κB kinase (IKK) subunit IKKβ. Here, we investigate whether small interfering RNA (siRNA) targeting IKKβ protects rats from renal ischemia- reperfusion injury in vivo. Renal ischemia-reperfusion injury was induced by clamping the renal artery for 45 min. Rats were treated before ischemia with IKKβ siRNA or scrambled siRNA, administered by renal artery injection. Treated animals were evaluated for renal IKKβ protein and mRNA expression, blood biochemistry, tissue histopathology, NF-κB/DNA binding activity, and expression of two downstream inflammatory cytokines, neutrophil gelatinase-associated lipocalin (NGAL) and IL-18. A local injection of IKKβ siRNA resulted in inhibition of renal IKKβ gene expression, NF-κB/DNA binding activity, and expression of NGAL and IL-18. Rats pretreated with IKKβ siRNA had significantly less blood urea nitrogen and serum creatinine levels and less renal tubular damage scores. Consequently, our data confirm that targeted silencing of IKKβ using siRNA substantially diminishes kidney injury and inflammation following ischemia-reperfusion.