Ly6/uPAR proteins regulate many essential functions in the nervous and immune systems and epithelium. Most of these proteins contain single β-structural LU domains with three protruding loops and are glycosylphosphatidylinositol (GPI)-anchored to a membrane. The GPI-anchor role is currently poorly studied. Here, we investigated the positional and orientational preferences of six GPI-anchored proteins in the receptor-unbound state by molecular dynamics simulations. Regardless of the linker length between the LU domain and GPI-anchor, the proteins interacted with the membrane by polypeptide parts and N-/O-glycans. Lynx1, Lynx2, Lypd6B, and Ly6H contacted the membrane by the loop regions responsible for interactions with nicotinic acetylcholine receptors, while Lypd6 and CD59 demonstrated unique orientations with accessible receptor-binding sites. Thus, GPI-anchoring does not guarantee an optimal ‘pre-orientation’ of the LU domain for the receptor interaction.