Thrombotic risk associated with chemotherapy including doxorubicin (DOX) has been frequently reported, yet the exact mechanism is not fully understood. Here, we report that DOX can induce procoagulant activity in platelets, an important contributor to thrombus formation. In human platelets, DOX increased phosphatidylserine (PS) exposure and PS-bearing microparticle generation. Consistently, DOX-treated platelets and generated microparticles induced thrombin generation, a representative marker for procoagulant activity. DOX-induced PS exposure appeared to be from intracellular Ca(2+) increase and ATP depletion which resulted in the activation of scramblase and inhibition of flippase. Along with this, apoptosis was induced by DOX as determined by the dissipation of mitochondrial membrane potential (Ψ), cytochrome c release, Bax translocation and caspase-3 activation. A Ca(2+) chelator (EGTA), caspase inhibitor (QVD) and antioxidants (Vitamin C and Trolox) can attenuate DOX-induced PS exposure and procoagulant activity significantly, suggesting that Ca(2+), apoptosis and Reactive oxygen species (ROS) were involved in DOX-enhanced procoagulant activity. Importantly, rat in vivo thrombosis model demonstrated that DOX could manifest prothrombotic effects through the mediation of platelet procoagulant activity which was accompanied by increased PS exposure and Ψ dissipation in platelets.