Glycosylphosphatidylinositols (GPIs) are complex glycolipids that are covalently linked to the C-terminus of proteins as a posttranslational modification. They anchor the attached protein to the cell membrane and are essential for normal functioning of eukaryotic cells. GPI-anchored proteins are structurally and functionally diverse. Many GPIs have been structurally characterized but comprehension of their biological functions, beyond the simple physical anchoring, remains largely speculative. Work on functional elucidation at a molecular level is still limited. This Review focuses on the roles of GPI unraveled by using synthetic molecules and summarizes the structural diversity of GPIs, as well as their biological and chemical syntheses.