Summary. Background: Prohibitins (PHBs), comprising the two homologous members PHB1 and PHB2, are ubiquitously expressed and highly conserved. The membrane PHBs have been reported to be involved in typhoid fever, obesity, and cancer metastasis. Proteomic studies have revealed the presence of PHBs in human platelets, but the roles of PHBs during platelet aggregation are unknown.Objectives: To investigate the role of PHBs in platelet aggregation. Methods and results: PHB1 and PHB2 were detected on the surfaces of human platelets by flow cytometry and confocal microscopy. The PHBs were distributed in lipid rafts, as determined by sucrose density centrifugation. In addition, the PHBs were associated with protease-activated receptor 1 (PAR1), as determined by Bm-TFF2 (a PAR1 agonist)-affinity chromatography, coimmunoprecipitation, and confocal microscopy. The platelet aggregation, αIIbβ3 activation, granular secretion and calcium mobilization stimulated by low concentrations of thrombin (0.05 U mL−1) or PAR1-activating peptide (PAR1-AP) (20 μm) were reduced or abolished as a result of the blockade of PHBs by anti-PHB antibodies or their Fab fragments; however, the same results were not obtained with induction by high concentrations of thrombin (0.6 U mL−1) or protease-activated receptor 4-activating peptide (300 μm). The calcium mobilization in MEG-01 megakaryocytes stimulated by PAR1-AP was significantly suppressed by PHB depletion with RNA interference against PHB1 and PHB2. Conclusions: PHBs are localized on the human platelet membrane and are involved in PAR1-mediated platelet aggregation. Until recently, PHBs were unknown as regulators of PAR1 signaling, and they may be effective targets for antiplatelet therapy.