Monascus yellow pigments (MYPs), as food colorants, are of great interest to the food industry, because of their beneficial biological activities. In this study, a comparative metabolomics strategy revealed the metabolic regulatory mechanism of MYP overproduction, comparing ammonium chloride with peptone as nitrogen sources. Metabolomics-based multivariate regression modeling showed that metabolic biomarkers/modules, such as glucose, lactate, and the pentose phosphate (PP) pathway, were closely associated with the biosynthesis of MYPs. Exogenous addition of glucose increased production of MYPs, whereas lactate reduced it. Inhibition of the PP pathway with dehydroepiandrosterone decreased MYP production, while increasing the shunting production of orange and red pigments. All these treatments significantly changed the expression profiles of the pigment biosynthetic gene cluster and the mycelial morphology. Overall, this study demonstrates the feasibility of elucidating the mechanism of MYP biosynthesis by comprehensive metabolomics analysis, as well as discovering potential engineering targets of efficiency improvements to commercial MYP production. Key points • Comparative metabolomics revealed the biomarkers/modules of MYP production. • A rational exogenously adding strategy was implemented to regulate MYP synthesis. • Expression profiles of gene cluster and mycelial morphology were characterized. Graphical abstract