The role for kidney TLR9 in ischemic acute kidney injury (AKI) remains unclear. In this study, we tested the hypothesis that renal proximal tubular TLR9 activation exacerbates ischemic AKI by promoting renal tubular epithelial apoptosis and inflammation. To test this hypothesis, we generated mice lacking TLR9 in renal proximal tubules (TLR9 fl/fl PEPCK Cre mice). Contrasting previous studies in global TLR9 knockout mice, mice lacking renal proximal tubular TLR9 were protected against renal ischemia/reperfusion (IR) injury, with reduced renal tubular necrosis, inflammation (decreased proinflammatory cytokine synthesis and neutrophil infiltration), and apoptosis (decreased DNA fragmentation and caspase activation) when compared with wild-type (TLR9 fl/fl) mice. Consistent with this, a selective TLR9 agonist oligonucleotide 1668 exacerbated renal IR injury in TLR9 fl/fl mice but not in renal proximal tubular TLR9-null mice. Furthermore, in cultured human and mouse proximal tubule cells, TLR9-selective ligands induced NF-κB activation, proinflammatory cytokine mRNA synthesis, as well as caspase activation. We further confirm in the present study that global TLR9 deficiency had no impact on murine ischemic AKI. Taken together, our studies show that renal proximal tubular TLR9 activation exacerbates ischemic AKI by promoting renal tubular inflammation, apoptosis as well as necrosis, after IR via NF-κB and caspase activation. Our studies further suggest the complex nature of TLR9 activation, as renal tubular epithelial TLR9 promotes cell injury and death whereas TLR9 signaling in other cell types may promote cytoprotective effects.