Understanding the causes of high-mannose (HM) glycosylation of recombinant IgG in CHO cells would facilitate the production of therapeutics. CHO cells grown with mannose as the major carbon source demonstrated a dramatic increase in total HM glycosylation in recombinant IgG, with no effect on cell growth, viability, or titer. Quantitative metabolomics and C-13 flux analysis were used to explore the mechanism for increased HM glycosylation and understand the metabolism of mannose in CHO cells. It was demonstrated that mannose was a good carbon source for CHO cell growth and IgG production, readily entering both glycolysis and the TCA Cycle. Previous mechanisms for increased HM glycosylation during antibody production have been attributed to changes in pH, osmolality, increased specific productivity, and nutrient limitation. The results from this study propose a novel mechanism where an increased carbon flux in the GDP-mannose synthetic pathway increased the intracellular concentration of mannose-containing metabolites. The abnormally high concentration of mannose and mannose-metabolites were shown to inhibit -mannosidase activity and it was proposed that this inhibition in the ER and Golgi caused the production of IgG with increased high-mannose glycosylation. Biotechnol. Bioeng. 2016;113: 1468-1480. (c) 2016 Wiley Periodicals, Inc.