The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib-IX, mediates platelet adhesion and activation. The cytoplasmic domains of the GPIb alpha and beta subunits contain binding sites for the phosphorylation-dependent signaling molecule, 14-3-3zeta. Here we show that a novel membrane-permeable inhibitor of 14-3-3zeta-GPIbalpha interaction, MPalphaC, potently inhibited VWF binding to platelets and VWF-mediated platelet adhesion under flow conditions. MPalphaC also inhibited VWF-dependent platelet agglutination induced by ristocetin. Furthermore, activation of the VWF binding function of GPIb-IX induced by GPIbbeta dephosphorylation is diminished by mutagenic disruption of the 14-3-3zeta binding site in the C-terminal domain of GPIbalpha, mimicking MPalphaC-induced inhibition, indicating that the inhibitory effect of MPalphaC is likely to be caused by disruption of 14-3-3zeta binding to GPIbalpha. These data suggest a novel 14-3-3zeta-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.