Purpose <p>Cancer cells rely on serine biosynthesis for growth, but its regulation in colorectal cancer (CRC) remains not well understood. This study identifies the m⁵C methyltransferase NSUN2 (NOP2/Sun domain family, member 2) as a key regulator of serine biosynthesis, revealing a novel mechanism driving CRC progression.</p> Methods <p>The expression and prognostic value of NSUN2 were evaluated using bioinformatics analyses and immunohistochemistry (IHC) assays. The effects of NSUN2 on cellular serine biosynthesis, intracellular reactive oxygen species (ROS) levels, and apoptosis levels were analyzed both in vitro and in vivo. Additionally, RNA sequencing, Methylated RNA Immunoprecipitation sequencing (MeRIP-seq), RNA immunoprecipitation (RIP), and RNA stability assays were utilized to screen and validate the association between NSUN2 and phosphoglycerate dehydrogenase (PHGDH).</p> Results <p>NSUN2 was found to be highly expressed in CRC and associated with poor patient survival. PHGDH, a direct downstream target of NSUN2, plays a crucial role in NSUN2-mediated serine biosynthesis. Furthermore, inhibition of NSUN2 significantly reduced the intracellular NADH/NAD⁺ and NADPH/NADP⁺ ratios, leading to an increase in ROS levels and apoptosis levels, thereby inhibiting CRC progression. Additionally, NSUN2 enhances PHGDH expression and mRNA stability by binding to the “reader” protein m⁵C-Aly/REF export factor (ALYREF).</p> Conclusions <p>This study identified a novel NSUN2/ALYREF/m⁵C-PHGDH axis might be promising therapeutic targets for CRC.</p>