Platelet adhesion and aggregation play a critical role in primary hemostasis. Uncontrolled platelet activation leads to pathologic thrombus formation and organ failure. The decisive central step for different processes of platelet activation is the increase in cytosolic Ca²⁺ activity ([Ca²⁺]_i). Activation-dependent depletion of intracellular Ca²⁺ stores triggers Ca²⁺ entry from the extracellular space. Stromal interaction molecule 1 (STIM1) has been identified as a Ca²⁺ sensor that regulates store-operated Ca²⁺ entry through activation of the pore-forming subunit Orai1, the major store-operated Ca²⁺ entry channel in platelets. In the present study, we show for the first time that the chaperone protein cyclophilin A (CyPA) acts as a Ca²⁺ modulator in platelets. CyPA deficiency strongly blunted activation-induced Ca²⁺ mobilization from intracellular stores and Ca²⁺ influx from the extracellular compartment and thus impaired platelet activation substantially. Furthermore, the phosphorylation of the Ca²⁺ sensor STIM1 was abrogated upon CyPA deficiency, as shown by immunoprecipitation studies. In a mouse model of arterial thrombosis, CyPA-deficient mice were protected against arterial thrombosis, whereas bleeding time was not affected. The results of the present study identified CyPA as an important Ca²⁺ regulator in platelets, a critical mechanism for arterial thrombosis.