Simultaneously to phospholipid flip-flop that supports the procoagulant activity of activated platelets, blebs, supported by actin reorganization, are formed at the plasma membrane and generate microvesicles. The molecular mechanism of microvesicle shedding from activated platelets implicates Ca influx and Ca-dependent protease, calpain. We previously demonstrated that the formation of lamellipodias and filopodias associated with platelet shape change involved the reorganization of actin filaments through a Cdc42/Rac1/p21-activated kinase (PAK)-dependent pathway. Here, we investigated whether platelet blebbing also depends on the Cdc42/Rac1/PAK pathway. Exposure of platelets in vitro to either a mixture of thrombin receptor-activating peptide (TRAP) and collagen or the Ca ionophore A23187 in the presence of Ca generates microvesicles that can be identified by flow cytometry. The calpain inhibitor, calpeptin, diminished microvesicle formation induced by the Ca ionophore A23187, confirming the role of calpain in this process. PAK1/2 is cleaved in a calpain-dependent manner, and calpeptin prevents this cleavage and allows a transient activation of the kinase. Inhibition of Cdc42 and Rac1 by toxin B from Clostridium difficile, that suppresses PAK1/2 activation induced by TRAP and collagen or by A23187 in the presence of calpeptin, decreases polymerization of actin, lamellipodia and filopodia formation and interferes with the shedding of microvesicles. We conclude that the Rac1/Cdc42/PAK pathway controls actin reorganization that is necessary for microvesicle shedding.