Detection of microbial components by immune cells via Toll-like receptors (TLRs) with subsequent induction of inflammation is essential for host defense. However, an overactive immune response can cause tissue damage and sepsis. The endogenous molecule hemoglobin and its derivative heme are often released into tissue compartments where there is infection in the presence of degrading blood. We found that hemoglobin synergizes with multiple TLR agonists to induce high levels of tumor necrosis factor and interleukin-6 from macrophages and that this synergy is independent of TLR4 and MyD88. In contrast, heme synergized with some but not all TLR agonists studied. Furthermore, the synergy of both hemoglobin and heme with lipopolysaccharide was suppressed by hemopexin, a plasma heme-binding protein. These studies suggest that hemoglobin and heme may substantially contribute to microbe-induced inflammation when bacterial or viral infection coexists with blood degradation and that hemopexin may play a role in controlling inflammation in such settings.