Rationale: The interaction between platelet glycoprotein (GP) Ib-IX and von Willebrand factor (VWF) is initiated by conformational changes in immobilized VWF and is also regulated by the intraplatelet proteins 14-3-3ζand filamin A. Both 14-3-3ζand filamin A associate with the cytoplasmic domain of GPIb, whereas little is known about their relationship in regulating the VWF binding function of GPIb-IX. Objective: To explore the mechanism underlying the roles of 14-3-3ζand filamin A in regulating the VWF binding function of GPIb-IX. Methods and Results: A truncation mutant of GPIbζ(△565) deleting the C-terminal 14-3-3ζbinding sites retained 14-3-3ζbinding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIb totally abolished 14-3-3ζbinding, indicating that the residues 551 to 564 of GPIbζare important in the interaction between 14-3-3ζ and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbζsuggesting phosphorylation of a population of GPIbζmolecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIb eliminated the association of 14-3-3ζwith △565. MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIbζ with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding, and diminished ability to mediate cell adhesion to VWF under flow conditions. Conclusions: These data suggest a phosphorylation-dependent binding of 14-3-3ζto central filamin A binding site of GPIbζ, and the dimeric 14-3-3ζ binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX.