GPI-APs are a class of lipid-anchored proteins expressed on the cell surface of eukaryotes. Their potential interaction with ordered lipid domains enriched in cholesterol and sphingolipids has been proposed to function in their intracellular transport. Here, we examined the importance of two saturated fatty acids within the phosphatidylinositol moiety of GPI-APs. These fatty acids are introduced by the action of lipid remodeling enzymes and required for the GPI-AP association within ordered lipid domains. The fatty acid remodeling is not required for both, efficient Golgi-to-plasma membrane transport and selective endocytosis via CLIC/GEEC pathway, whereas cholesterol depletion significantly affects both pathways independent of their fatty acid structure. Therefore, the mechanism of cholesterol dependence seems not related to the potential interaction with ordered lipid domain mediated by two saturated fatty acids.