Ischemia/reperfusion injury (IRI) can activate innate immunity through the engagement of Toll-like receptors (TLRs) by endogenous ligands. TLR4, expressed in the kidney, is a potential mediator of innate activation and inflammation. Using a mouse model of kidney IRI, the authors demonstrated a significant increase in TLR4 expression by tubular epithelial cells (TECs) and infiltrating leukocytes within the kidney following ischemia. TLR4 signaling through the MyD88-dependent pathway was required for the full development of kidney IRI, as both *TLR4*^−/−^ and *MyD88*^−/−^ mice were protected against kidney dysfunction, tubular damage, neutrophil and macrophage accumulation, and expression of proinflammatory cytokines and chemokines. In vitro, WT kidney TECs produced proinflammatory cytokines and chemokines and underwent apoptosis after ischemia, which were attenuated in *TLR4*^−/−^ and *MyD88*^−/−^ TECs. Additionally, upregulation of endogenous ligands such as high-mobility group box 1 (HMGB1), hyaluronan, and biglycan was observed, suggesting that these ligands may be the source of TLR4 activation. To determine the relative contribution of TLR4 expression by parenchymal cells or leukocytes to kidney damage during IRI, chimeric mice were generated. *TLR4*^−/−^ mice engrafted with WT hematopoietic cells had significantly lower serum creatinine and less tubular damage than WT mice reconstituted with *TLR4*^−/−^ bone marrow (BM), indicating that TLR4 signaling in intrinsic kidney cells plays a dominant role in mediating kidney damage.Ischemia/reperfusion injury (IRI) can activate innate immunity through the engagement of Toll-like receptors (TLRs) by endogenous ligands. TLR4, expressed in the kidney, is a potential mediator of innate activation and inflammation. Using a mouse model of kidney IRI, the authors demonstrated a significant increase in TLR4 expression by tubular epithelial cells (TECs) and infiltrating leukocytes within the kidney following ischemia. TLR4 signaling through the MyD88-dependent pathway was required for the full development of kidney IRI, as both *TLR4*^−/−^ and *MyD88*^−/−^ mice were protected against kidney dysfunction, tubular damage, neutrophil and macrophage accumulation, and expression of proinflammatory cytokines and chemokines. In vitro, WT kidney TECs produced proinflammatory cytokines and chemokines and underwent apoptosis after ischemia, which were attenuated in *TLR4*^−/−^ and *MyD88*^−/−^ TECs. Additionally, upregulation of endogenous ligands such as high-mobility group box 1 (HMGB1), hyaluronan, and biglycan was observed, suggesting that these ligands may be the source of TLR4 activation. To determine the relative contribution of TLR4 expression by parenchymal cells or leukocytes to kidney damage during IRI, chimeric mice were generated. *TLR4*^−/−^ mice engrafted with WT hematopoietic cells had significantly lower serum creatinine and less tubular damage than WT mice reconstituted with *TLR4*^−/−^ bone marrow (BM), indicating that TLR4 signaling in intrinsic kidney cells plays a dominant role in mediating kidney damage.