The study investigates the role of Y-box-binding protein 1 (YBX1) in esophageal squamous cell carcinoma (ESCC) and its mechanism of action. YBX1 is frequently upregulated in ESCC tissues compared to normal tissues, and gain- and loss-of-function assays show that YBX1 promotes the proliferation and metastasis of ESCC cells both in vitro and in vivo. The study identifies NOP2/Sun RNA methyltransferase family member 2 (NSUN2) as a critical RNA methyltransferase that facilitates YBX1-mediated ESCC progression. Mechanistically, YBX1 binds to and stabilizes the mRNA of spermine oxidase (SMOX), a target gene containing an m5C site in its coding sequence (CDS) region, which coincides with the binding site of YBX1. Overexpression of SMOX-WT but not SMOX-Mut partially restores the proliferation and invasion ability of ESCC cells curbed by YBX1 knockdown. Additionally, YBX1 activates the mTORC1 signaling pathway by stabilizing SMOX mRNA. The study concludes that YBX1 promotes ESCC development by stabilizing SMOX mRNA in an m5C-dependent manner, providing a valuable therapeutic target for ESCC.The study investigates the role of Y-box-binding protein 1 (YBX1) in esophageal squamous cell carcinoma (ESCC) and its mechanism of action. YBX1 is frequently upregulated in ESCC tissues compared to normal tissues, and gain- and loss-of-function assays show that YBX1 promotes the proliferation and metastasis of ESCC cells both in vitro and in vivo. The study identifies NOP2/Sun RNA methyltransferase family member 2 (NSUN2) as a critical RNA methyltransferase that facilitates YBX1-mediated ESCC progression. Mechanistically, YBX1 binds to and stabilizes the mRNA of spermine oxidase (SMOX), a target gene containing an m5C site in its coding sequence (CDS) region, which coincides with the binding site of YBX1. Overexpression of SMOX-WT but not SMOX-Mut partially restores the proliferation and invasion ability of ESCC cells curbed by YBX1 knockdown. Additionally, YBX1 activates the mTORC1 signaling pathway by stabilizing SMOX mRNA. The study concludes that YBX1 promotes ESCC development by stabilizing SMOX mRNA in an m5C-dependent manner, providing a valuable therapeutic target for ESCC.