The hemoglobin scavenger receptor (HbSR) CD163 is a monocyte/macrophage-specific glycoprotein that binds and facilitates uptake of haptoglobin-hemoglobin (Hp-Hb) complexes, which are rapidly formed in the circulation upon hemolysis of red blood cells. Hemolysis can be caused by a diverse range of infectious agents and provides pathogens a source of iron to enhance their survival and replication. Previous work demonstrated that lipopolysaccharide (LPS) activates monocytes to cleave cell-bound HbSR into a soluble mediator that retains the capacity to bind Hp-Hb complexes. We report that blocking LPS activation of Toll-like receptor 4 prevents LPS-mediated shedding of CD163. Furthermore, activation of two other cell surface Toll-like receptors (TLR), TLR2 and TLR5, induces shedding of the HbSR from human monocytes. In contrast, treatment of monocytes with intracellular TLR3, TLR7, and TLR9 agonists failed to cause HbSR shedding, suggesting that this shedding event is selective to cell surface TLR activation. These data demonstrate that the soluble HbSR is released from monocytic cells in response to TLR signaling as an acute innate immune response to extracellular pathogen infections.