Eukaryotic cells are coated with an abundance of glycosylphosphatidylinositol anchor proteins (GPI-APs) that play crucial roles in fertilization, neurogenesis, and immunity. Covalent addition of structurally diverse GPI anchorages at the carboxyl termini of target proteins is catalyzed by an endoplasmic reticulum integral membrane GPI transamidase complex (GPI-T) conserved among all eukaryotes. Despite its important role in developmental and cancer biology, a detailed picture of this intricate multi-component machinery remains elusive. Here, we report the cryo-electron microscopy (EM) structure of the human GPI-T complex at a global 2.53 Å resolution, revealing an assembly mechanism whereby the catalytic subunit PIGK is optimally positioned to accommodate its characteristic amphipathic substrates. Further, structural and functional characterizations suggest a previously unrecognized composite GPI-binding site formed by PIGU/PIGK/PIGT subunits and rationalize genetic diseases associated with GPI-T point mutations. Our work presents an important step towards the mechanistic understanding of GPI-AP biosynthesis.