Monascus pigments (MPs) are widely used natural colorants in Asian countries. MPs production can be significantly enhanced by glycerol, though its mechanism remains unknown. In this study, behind glycerol-enhanced MPs production was investigated through physiological and transcriptome analyses. Monascus pilosus MS-1 was cultured in synthetic media with 0, 40 or 160 g/L glycerol which were marked as CK, G40, and G160, respectively. Monascus growth and MPs production were significantly enhanced by glycerol. Differentially expressed genes (DEGs) found in the CK vs G40 group involved in oxidation-reduction and glycolytic processes were upregulated, while that of RNA-DNA hybrid ribonuclease activity and RNA-dependent DNA biosynthetic process were downregulated. The downregulated genes related to G160 were significantly enriched in GO terms of nucleolus, 90S preribosome, and small and large subunit precursors. KEGG analysis indicated that most of the upregulated genes belonging to the CK vs G40 and CK vs G160 groups were related to glycolysis and carbon metabolism. However, DEGs belonging to groups related to G160 involved in ribosome biogenesis in eukaryotes and pyrimidine metabolism were downregulated. Therefore, glycerol promotion of MPs production may be attributed to more substrates for MPs biosynthesis generated from glycolysis and carbon metabolism.