Glycine betaine (GB) serves as both a methyl donor and osmoprotectant in microorganisms, facilitating growth and enhancing metabolic product yields. While the polyketide metabolites from Monascus purpureus, such as Monascus pigments (MPs) and monacolin K (MK), have been extensively studied, the effects of GB on their production and the underlying molecular mechanisms remain insufficiently explored. In this study, various concentrations of GB were added to Monascus purpureus M1 cultures, followed by RNA sequencing, RT-qPCR, differential gene expression analysis, and functional enrichment to investigate the regulatory impact of GB on polyketide metabolism. Protein–protein interaction network analysis identified key upregulated genes, including RPS15, RPS14, RPS5, NDK1, EGD2, and ATP9, particularly during the later growth phases. GB significantly upregulated genes involved in stress response, secondary metabolism, and polyketide biosynthesis. Scanning electron microscopy, HPLC, and UV-Vis spectrophotometry further confirmed that GB promoted both strain growth and polyketide production, with red pigment and MK production increasing by 120.08% and 93.4%, respectively. These results indicate that GB enhances growth and polyketide metabolism in Monascus purpureus by functioning as both a methyl donor and osmoprotectant, offering new insights into optimizing microbial polyketide production and revealing gene regulatory mechanisms by GB in Monascus purpureus.