Mannosylation in the endoplasmic reticulum is a key process for synthesizing various glycans. Guanosine diphosphate mannose (GDP-Man) and dolichol phosphate-mannose serve as donor substrates for mannosylation in mammals and are used in N-glycosylation, O-mannosylation, C-mannosylation, and the synthesis of glycosylphosphatidylinositol-anchor (GPI-anchor). Here, we report for the first time that low-abundant uridine diphosphate-mannose (UDP-Man), which can serve as potential donor substrate, exists in mammals. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that mouse brain, especially hypothalamus and neocortex, contains higher concentrations of UDP-Man compared to other organs. In cultured human cell lines, addition of mannose in media increased UDP-Man concentrations in a dose-dependent manner. These findings indicate that in mammals the minor nucleotide sugar UDP-Man regulates glycosylation, especially mannosylation in specific organs or conditions.