Hemolysis, a pathological component of many diseases, is associated with thrombosis and vascular dysfunction. Hemolytic products, including cell-free hemoglobin and free heme directly activate platelets. However, the effect of hemolysis on platelet degranulation, a central process in not only thrombosis, but also inflammatory and mitogenic signaling, remains less clear. Our group showed that hemoglobin-induced platelet activation involved the production of mitochondrial reactive oxygen species (mtROS). However, the molecular mechanism by which extracellular hemolysis induces platelet mtROS production, and whether these mtROS regulate platelet degranulation remains unknown. Here, we demonstrate using isolated human platelets that cell free heme is a more potent agonist for platelet activation than hemoglobin, and stimulates the release of a specific set of molecules, including the glycoprotein thrombospondin-1 (TSP-1), from the α-granule of platelets. We uncover the mechanism of heme-mediated platelet mtROS production which is dependent on the activation of platelet toll-like receptor 4 (TLR4) signaling and leads to the downstream phosphorylation and inhibition of complex-V by the serine kinase Akt. Notably, inhibition of platelet TLR4 or Akt, or scavenging of mtROS prevents heme-induced granule release in vitro. Further, heme-dependent granule release is significantly attenuated in vivo in mice lacking TLR4 or those treated with the mtROS scavenger MitoTEMPO. These data elucidate a novel mechanism of TLR4-mediated mitochondrial regulation, establish the mechanistic link between hemolysis and platelet degranulation, and begin to define the heme and mtROS-dependent platelet secretome. These data have implications for hemolysis-induced thrombo-inflammatory signaling and for the consideration of platelet mitochondria as a therapeutic target in hemolytic disorders.