Destabilized heme proteins release heme, and free heme is toxic. Heme is now recognized as an agonist for the TLR4 receptor. This study examined whether the TLR4 receptor mediates the nephrotoxicity of heme, specifically, the effects of heme on renal blood flow and inflammatory responses. We blocked TLR4 signaling by the specific antagonist TAK-242. Intravenous administration of heme to mice promptly reduced renal blood flow, an effect attenuated by TAK-242. In vitro, TAK-242 reduced heme-elicited activation of NF-B and its downstream gene MCP-1; in contrast, TAK-242 failed to reduce heme-induced activation of the anti-inflammatory transcription factor, Nrf2, and its downstream gene HO-1. TAK-242 did not reduce heme-induced renal MCP-1 upregulation in vivo. TAK-242 did not reduce dysfunction and histologic injury in the glycerol model of heme protein-induced acute kidney injury (AKI), findings corroborated by studies in TLR4+/+ and TLR4-/- mice. We conclude that: 1) acute heme-mediated renal vasoconstriction occurs through TLR4 signaling; 2) proinflammatory effects of heme in renal epithelial cells involve TLR4 signaling, whereas the anti-inflammatory effects of heme do not; 3) TLR4 signaling does not mediate the proinflammatory effects of heme in the kidney; 4) major mechanisms underlying glycerol-induced, heme protein-mediated AKI do not involve TLR4 signaling. These findings in the glycerol model are in stark contrast with findings in all other AKI models studied to-date and emphasize the importance of TLR4-independent pathways of heme protein-mediated injury in this model. Finally, these studies urge caution when using observations derived in vitro to predict what may occur in vivo.