The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib–IX, mediates platelet adhesion and activation. The cytoplasmic domains of the GPIb α and β subunits contain binding sites for the phosphorylation-dependent signaling molecule, 14-3-3. Here we show that a novel membrane-permeable inhibitor of 14-3-3-GPIbα interaction, MPα C, potently inhibited VWF binding to platelets and VWF-mediated platelet adhesion under flow conditions. MPαC also inhibited VWF-dependent platelet agglutination induced by ristocetin. Furthermore, activation of the VWF binding function of GPIb-IX induced by GPIbβ dephosphorylation is diminished by mutagenic disruption of the 14-3-3 binding site in the C-terminal domain of GPIbα, mimicking MPα C-induced inhibition, indicating that the inhibitory effect of MPαC is likely to be caused by disruption of 14-3-3 binding to GPIbα. These data suggest a novel 14-3-3-dependent regulatory mechanism that controls the VWF binding function of GPIb-IX, and also suggest a new type of antiplatelet agent that may be potentially useful in preventing or treating thrombosis.