Dolichol-phosphate-mannose (DPM) synthase generates mannosyl donors for glycosylphosphatidylinositols, N-glycan and protein O- and C-mannosylation. In Saccharomyces cerevisiae, this enzyme is encoded by DPM1. We reported previously that mammalian DPM synthase contains catalytic DPM1 and regulatory DPM2 subunits, and that DPM1 requires DPM2 for its stable expression in the endoplasmic reticulum. Here we report that human DPM synthase consists of three subunits. The third subunit, DPM3, comprises 92 amino acids associated with DPM1 via its C-terminal domain and with DPM2 via its N-terminal portion. The stability of DPM3 was dependent upon DPM2. However, overexpression of DPM3 in Lec15 cells, a null mutant of DPM2, restored the biosynthesis of DPM with an increase in DPM1, indicating that DPM3 directly stabilized DPM1. Therefore, DPM2 stabilizes DPM3 and DPM3 stabilizes DPM1. DPM synthase activity was 10 times higher in the presence of DPM2, indicating that DPM2 also plays a role in the enzymatic reaction. Schizosaccharomyces pombe has proteins that resemble three human subunits; S.pombe DPM3 restored biosynthesis of DPM in Lec15 cells, indicating its orthologous relationship to human DPM3.