Glucose is a critical nutrient for cell proliferation. However, the molecular pathways that regulate glucose metabolism are still elusive. We discovered that co-activator-associated arginine methyltransferase 1 (CARM1) suppresses glucose metabolism toward serine biosynthesis. By tracing the ¹³C-labeled glucose, we found that Carm1 knockout mouse embryonic fibroblasts exhibit significantly increased de novo serine synthesis than WT cells. This is caused, at least in part, by the reduced pyruvate kinase (PK) activity in these cells. The M2 isoform of PK (PKM2) is arginine-methylated by CARM1, and methylation enhances its activity. Mechanistically, CARM1 methylates PKM2 at arginines 445 and 447, which enhances PKM2 tetramer formation. Consequently, Carm1 knockout cells exhibit significant survival advantages over WT cells when extracellular serine is limited, likely due to their enhanced de novo serine synthesis capacity. Altogether, we identified CARM1 as an important regulator of glucose metabolism and serine synthesis.