Platelets (PLT) are blood cells that are formed by the release of parts of the cytoplasm of mature megakaryocytes. In the circulation, they are in their resting state, but upon vessel wall injury, they activate, which leads to PLT shape change and the formation of a thrombus. Actin polymerization occurs as a result of PLT activation, and PLT adhesion is achieved by the formation of adhesive extensions: filopodia and lamellipodia. Membrane remodeling and vesicular trafficking are needed for PLT activation, and these processes are mediated also by phosphoinositides (PIs), phosphorylated membrane lipids. Two most abundant PIs in the cells are phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-monophosphate (PI4P), the later being a major source for PI(4,5)P2. PI4P can be produced by type II phosphatidylinositol 4-kinases (PI4KIIα and PI4KIIβ) and type III kinases (PI4KIIIα and PI4KIIIβ). In this study, we show that specific pharmacological inhibition of PI4KIIIβ, with inhibitor IN-9, reduces PLT spreading on collagen. Inhibited platelets are more rounded and unexpanded, exhibit an inability to polymerize F-actin as evidenced by an increased number of filopodia compared to lamellipodia, and the absence of actinomyosin stress fibers that help maintain spreaded platelet. Furthermore, we showed the localization of PI4KIIα in resting and activated platelets and its colocalization with α-tubulin using immunofluorescence. We also show that inhibition of PI4KIIα with PI-273 mildly only at higher concentrations reduces platelet spreading on collagen. indicating a weaker role of PI4KIIα in this aspect of platelet function.