Hemolytic or hemorrhagic episodes are often associated with inflammation even when infectious agents are absent suggesting that red blood cells (RBCs) release damage-associated molecular patterns (DAMPs). DAMPs activate immune and nonimmune cells through pattern recognition receptors. Heme, released from RBCs, is a DAMP and induces IL-1β production through the activation of the nucleotide-binding domain and leucine-rich repeat-containing family and pyrin domain containing 3 (NLRP3) in macrophages; however, other cellular targets of heme-mediated inflammasome activation were not investigated. Because of their location, endothelial cells can be largely exposed to RBC-derived DAMPs; therefore, we investigated whether heme and other hemoglobin- (Hb-) derived species induce NLRP3 inflammasome activation in these cells. We found that heme upregulated NLRP3 expression and induced active IL-1β production in human umbilical vein endothelial cells (HUVECs). LPS priming largely amplified the heme-mediated production of IL-1β. Heme administration into C57BL/6 mice induced caspase-1 activation and cleavage of IL-1β which was not observed in NLRP3^−/− mice. Unfettered production of reactive oxygen species played a critical role in heme-mediated NLRP3 activation. Activation of NLRP3 by heme required structural integrity of the heme molecule, as neither protoporphyrin IX nor iron-induced IL-1β production. Neither naive nor oxidized forms of Hb were able to induce IL-1β production in HUVECs. Our results identified endothelial cells as a target of heme-mediated NLRP3 activation that can contribute to the inflammation triggered by sterile hemolysis. Thus, understanding the characteristics and cellular counterparts of RBC-derived DAMPs might allow us to identify new therapeutic targets for hemolytic diseases.