In eukaryotic cells, phosphatidylserine (PS) is synthesized in the endoplasmic reticulum (ER) but is highly enriched in the plasma membrane (PM), where it contributes negative charge and to specific recruitment of signaling proteins. This distribution relies on transport mechanisms whose nature remains elusive. Here, we found that the PS transporter Osh6p extracted phosphatidylinositol 4-phosphate (PI4P) and exchanged PS for PI4P between two membranes. We solved the crystal structure of Osh6p: PI4P complex and demonstrated that the transport of PS by Osh6p depends on PI4P recognition in vivo. Finally, we showed that the PI4P-phosphatase Sac1p, by maintaining a PI4P gradient at the ER/PM interface, drove PS transport. Thus, PS transport by oxysterol-binding protein-related protein (ORP)/oxysterol-binding homology (Osh) proteins is fueled by PI4P metabolism through PS/PI4P exchange cycles.