Von Willebrand factor (VWF) secreted by Weibel-Palade bodies within endothelial cells is critical to maintain normal platelet adhesion during vascular arrest. Because the transportation of VWF is microtubule-dependent partly via kinesin activity, suppression of kinesin could affect intracellular trafficking of VWF. In this study, we investigated the role of Kif5b, the key member of the kinesin superfamily, in the processing and secretion of VWF. A hypothetical interaction between VWF and Kif5b was confirmed and the tail domain of Kif5b was identified as VWF binding region. Knocking-down Kif5b in human umbilical vein endothelial cells led to significantly increased non-stimulated VWF secretion, considerably higher ratio of pro-VWF to mature VWF, and shorter VWF length. Consistent to the in vitro assay, Kif5b knockdown mice demonstrated dramatically increased VWF secretion after epinephrine stimulation. Significantly prolonged bleeding time was observed in these Kif5b knockdown mice as well, which was further elucidated by the decreased basal/regulated VWF secretion in Kif5b knockdown mice comparing to their wild-type littermates. Taken together, these findings suggest that Kif5b modulates processing and secretion of VWF in vitro and in vivo.