Toll-like receptors (TLRs) can detect endogenous danger molecules released upon tissue injury resulting in the induction of a proinflammatory response. One of the TLR family members, TLR4, is constitutively expressed at RNA level on renal epithelium and this expression is enhanced upon renal ischemia/reperfusion (I/R) injury. The functional relevance of this organ-specific upregulation remains however unknown. We therefore investigated the specific role of TLR4 and the relative contribution of its two downstream signaling cascades, the MyD88-dependent and TRIF-dependent cascades in renal damage by using TLR4−/−, MyD88−/− and TRIF-mutant mice that were subjected to renal ischemia/reperfusion injury. Our results show that TLR4 initiates an exaggerated proinflammatory response upon I/R injury, as reflected by lower levels of chemokines and infiltrating granulocytes, less renal damage and a more preserved renal function in TLR4−/− mice as compared to wild type mice. In vitro studies demonstrate that renal tubular epithelial cells can coordinate an immune response to ischemic injury in a TLR4-dependent manner. In vivo we found that epithelial- and leukocyte-associated functional TLR4 contribute in a similar proportion to renal dysfunction and injury as assessed by bone marrow chimeric mice. Surprisingly, no significant differences were found in renal function and inflammation in MyD88−/− and TRIF-mutant mice compared with their wild types, suggesting that selective targeting of TLR4 directly may be more effective for the development of therapeutic tools to prevent I/R injury than targeting the intracellular pathways used by TLR4. In conclusion, we identified TLR4 as a cellular sentinel for acute renal damage that subsequently controls the induction of an innate immune response.