Serine and glycine are biosynthetically linked and play crucial roles in the synthesis of proteins, nucleic acids, and lipids, which are essential for cancer cell growth. They also affect cellular antioxidative capacity, supporting tumor homeostasis. The glycine cleavage system, which refuels one-carbon metabolism, is a key pathway in serine/glycine biosynthesis. Genetic and functional evidence indicates that hyperactivation of the serine/glycine biosynthetic pathway drives oncogenesis. Recent advancements in understanding these pathways offer new opportunities for drug development, dietary intervention, and biomarker identification in cancer treatment. Serine biosynthesis is a central hub in cancer metabolism, with glycolysis providing the initial glucose source. The serine biosynthetic pathway diverges from glycolysis, converting 3-phosphoglycerate to serine through a series of enzymatic reactions. Phosphoglycerate dehydrogenase (PHGDH) and NAD are key enzymes in this process, and their upregulation is often observed in triple-negative breast cancer and melanoma. Suppression of PHGDH inhibits cell proliferation, suggesting that additional outputs of serine biosynthesis contribute to its importance in cancer. Serine and glycine are also involved in one-carbon metabolism, which is essential for the synthesis of nucleotides, purines, and DNA methylation. The conversion of serine to glycine by serine hydroxymethyltransferase (SHMT) is a critical step in this pathway. SHMT1 and SHMT2 are transcriptional targets of c-Myc, and their expression changes in various tumors. Glycine is also important for maintaining cellular redox balance and supporting oxidative phosphorylation. Targeting serine and glycine metabolism, particularly through the inhibition of SHMT, may represent a novel therapeutic strategy for cancer treatment.Serine and glycine are biosynthetically linked and play crucial roles in the synthesis of proteins, nucleic acids, and lipids, which are essential for cancer cell growth. They also affect cellular antioxidative capacity, supporting tumor homeostasis. The glycine cleavage system, which refuels one-carbon metabolism, is a key pathway in serine/glycine biosynthesis. Genetic and functional evidence indicates that hyperactivation of the serine/glycine biosynthetic pathway drives oncogenesis. Recent advancements in understanding these pathways offer new opportunities for drug development, dietary intervention, and biomarker identification in cancer treatment. Serine biosynthesis is a central hub in cancer metabolism, with glycolysis providing the initial glucose source. The serine biosynthetic pathway diverges from glycolysis, converting 3-phosphoglycerate to serine through a series of enzymatic reactions. Phosphoglycerate dehydrogenase (PHGDH) and NAD are key enzymes in this process, and their upregulation is often observed in triple-negative breast cancer and melanoma. Suppression of PHGDH inhibits cell proliferation, suggesting that additional outputs of serine biosynthesis contribute to its importance in cancer. Serine and glycine are also involved in one-carbon metabolism, which is essential for the synthesis of nucleotides, purines, and DNA methylation. The conversion of serine to glycine by serine hydroxymethyltransferase (SHMT) is a critical step in this pathway. SHMT1 and SHMT2 are transcriptional targets of c-Myc, and their expression changes in various tumors. Glycine is also important for maintaining cellular redox balance and supporting oxidative phosphorylation. Targeting serine and glycine metabolism, particularly through the inhibition of SHMT, may represent a novel therapeutic strategy for cancer treatment.