Although the genes involved in the biosynthesis of glycosylphosphatidylinositol (GPI) are well characterized, the regulation of GPI biosynthesis remains unclear. We isolated and characterized a mutant cell line showing decreased surface expression of CD59 and the accumulation of GPI intermediates. The mutant cell line was partially defective in MPDU1, which encodes a protein required for the utilization of dolichol-phosphate mannose. Overexpression of PIGV, which encodes GPI mannosyltransferase II, restored the surface expression of CD59 and normalized the accumulation of GPI intermediates in the mutant cells. Among all known genes involved in GPI biosynthetic pathway, only PIGV had such suppressive activity. PIGV, however, did not restore the abnormality of N-glycosylation caused by MPDU1 mutation. Our results suggest that GPI mannosyltransferase II is the rate-limiting enzyme in GPI biosynthesis under limited dolichol-phosphate mannose availability.