VASP plays crucial roles in controlling F-actin-driven processes and growing evidence indicates that VASP function is modulated by phosphorylation at multiple sites. However, the complexity of mammalian system prevents the clear understanding of the role of VASP phosphorylation. In this study, we took advantage of Dictyostelium which possesses only one member of the Ena/VASP family to investigate the functional roles of VASP phosphorylation. Our results demonstrated that hyperosmotic stress and cAMP stimulation cause VASP phosphorylation. VASP phosphorylation plays a negative role for the early steps of filopodia/microspikes formation. VASP phosphorylation appears to modulate VASP localization at the membrane cortex and its interactions with WASP and WIPa. Analysis of chemotaxis of cells expressing VASP mutants showed that VASP phosphorylation is required for the establishment of cell polarity under a cAMP gradient.