The interaction of platelet glycoprotein (GP) Ib-IX with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture initiates platelet transient adhesion to the injured vessel wall, which triggers intracellular signaling cascades leading to platelet activation and thrombus formation. 14-3-3 zeta has been verified to regulate the VWF binding function of GPIb-IX by interacting with the cytoplasmic domains of GPIb-IX. However, the data regarding the role of 14-3-3 zeta in GPIb-IX-VWF interaction-induced signaling still remain controversial. In the present study, the data indicate that the S609A mutation replacing Ser(609) of GPIb alpha with alanine (S609A) significantly prevented the association of 14-3-3 zeta with GPIba before and after the VWF binding to GPIb alpha. GPIb-IX-VWF interaction-induced activations of Src family kinases and protein kinase C were clearly reduced in S609A mutation. Furthermore, S609A mutation significantly inhibited GPIb-IX-VWF interaction-induced elevation of cytoplasmic Ca2+ levels in flow cytometry analysis. Taken together, these data indicate that the association of 14-3-3 zeta with the cytoplasmic domain of GPIb alpha plays an important role in GPIb-IX-VWF interaction-induced signaling.