Abstract: GPI7 is involved in adding ethanolaminephosphate to the second mannose in the biosynthesis of glycosylphosphatidylinositol (GPI) in Saccharomyces cerevisiae. We isolated gpi7 mutants, which have defects in cell separation and a daughter cell-specific growth defect at the non-permissive temperature. WSC1, RHO2, ROM2, GFA1, and CDC5 genes were isolated as multicopy suppressors of gpi7-2 mutant. Multicopy suppressors could suppress the growth defect of gpi7 mutants but not the cell separation defect. Loss of function mutations of genes involved in the Cbk1p-Ace2p pathway, which activates the expression of daughter-specific genes for cell separation after cytokinesis, bypassed the temperature-sensitive growth defect of gpi7 mutants. Furthermore, deletion of EGT2, one of the genes controlled by Ace2p and encoding a GPI-anchored protein required for cell separation, ameliorated the temperature sensitivity of the gpi7 mutant. In this mutant, Egt2p was displaced from the septal region to the cell cortex, indicating that GPI7 plays an important role in cell separation via the GPI-based modification of daughter-specific proteins in S. cerevisiae.