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In addition to the relevance of heme\u2010regulated proteins from the complement and blood coagulation systems, activation of the TLR4 signalling pathway by heme was ascribed a crucial role in the progression of these pathological processes. Heme binding to the TLR4\u2010MD2 complex has been proposed recently, however, essential mechanistic information of the processes at the molecular level, such as heme\u2010binding kinetics, the heme\u2010binding capacity and the respective heme\u2010binding sites (HBMs) is still missing. We report the interaction of TLR4, MD2 and the TLR4\u2010MD2 complex with heme and the consequences thereof by employing biochemical, spectroscopic, bioinformatic and physiologically relevant approaches. Heme binding occurs transiently through interaction with up to four HBMs in TLR4, two HBMs in MD2 and at least four HBMs in their complex. Functional studies highlight that mutations of individual HBMs in TLR4 preserve full receptor activation by heme, suggesting that heme interacts with TLR4 through different binding sites independently of MD2. 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