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.