Aims: In hemolysis, which is accompanied by increased levels of labile redox-active heme and is often associated with hemostatic abnormalities, a decreased activity of activated protein C (APC) is routinely detected. APC is a versatile enzyme that exerts its anticoagulant function through inactivation of clotting factors Va and VIIIa. APC has not been demonstrated to be affected by heme as described for other clotting factors and, thus, is a subject of investigation. Results: We report the interaction of heme with APC and its impact on the protein function by employing spectroscopic and physiologically relevant methods. Binding of heme to APC results in inhibition of its amidolytic and anticoagulant activity, increase of the peroxidase-like activity of heme, and protection of human umbilical vein endothelial cells from heme-induced hyperpermeability. To define the sites that are responsible for heme binding, we mapped the surface of APC for potential heme-binding motifs. T285GWGYHSSR293 and W387IHGHIRDK395, both located on the basic exosite, turned out as potential heme-binding sites. Molecular docking employing a homology model of full-length APC indicated Tyr289 and His391 as the Fe(III)-coordinating amino acids. Innovation: The results strongly suggest that hemolysis-derived heme may directly influence the protein C pathway through binding to APC, conceivably explaining the decreased activity of APC under hemolytic conditions. Further, these results extend our understanding of heme as a multifaceted effector molecule within coagulation and may allow for an improved understanding of disease development in hemostasis under hemolytic conditions. Conclusion: Our study identifies APC as a heme-binding protein and provides insights into the functional consequences.