RNA 5-methylcytosine writer NSUN5 promotes hepatocellular carcinoma (HCC) cell proliferation via a ZBED3-dependent mechanism. This study reveals that NSUN5, a conserved RNA 5-methylcytosine methyltransferase, is upregulated in HCC tissues and correlates with poor clinical outcomes. NSUN5 knockdown inhibits, while overexpression promotes, HCC cell proliferation. Using methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA immunoprecipitation sequencing (RIP-seq), the study identifies ZBED3 as a novel downstream target of NSUN5. ZBED3 overexpression counteracts the tumor-suppressing effect of NSUN5 knockdown and restores the Wnt/β-catenin signaling pathway. These findings demonstrate that NSUN5 promotes HCC development through the ZBED3/Wnt/β-catenin signaling pathway, offering a potential therapeutic target for HCC treatment. The study also highlights the role of NSUN5 in HCC progression, with elevated NSUN5 expression associated with increased tumor grade, stage, and TP53 mutation status. NSUN5 knockdown significantly reduces ZBED3 expression and inhibits HCC cell proliferation, while NSUN5 overexpression enhances ZBED3 expression and promotes cell growth. The results indicate that NSUN5 functions as an oncogene in HCC by modulating ZBED3 through m5C modification, thereby driving tumor progression. The study provides insights into the molecular mechanisms underlying NSUN5-mediated HCC development and identifies ZBED3 as a key downstream effector in this process.RNA 5-methylcytosine writer NSUN5 promotes hepatocellular carcinoma (HCC) cell proliferation via a ZBED3-dependent mechanism. This study reveals that NSUN5, a conserved RNA 5-methylcytosine methyltransferase, is upregulated in HCC tissues and correlates with poor clinical outcomes. NSUN5 knockdown inhibits, while overexpression promotes, HCC cell proliferation. Using methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA immunoprecipitation sequencing (RIP-seq), the study identifies ZBED3 as a novel downstream target of NSUN5. ZBED3 overexpression counteracts the tumor-suppressing effect of NSUN5 knockdown and restores the Wnt/β-catenin signaling pathway. These findings demonstrate that NSUN5 promotes HCC development through the ZBED3/Wnt/β-catenin signaling pathway, offering a potential therapeutic target for HCC treatment. The study also highlights the role of NSUN5 in HCC progression, with elevated NSUN5 expression associated with increased tumor grade, stage, and TP53 mutation status. NSUN5 knockdown significantly reduces ZBED3 expression and inhibits HCC cell proliferation, while NSUN5 overexpression enhances ZBED3 expression and promotes cell growth. The results indicate that NSUN5 functions as an oncogene in HCC by modulating ZBED3 through m5C modification, thereby driving tumor progression. The study provides insights into the molecular mechanisms underlying NSUN5-mediated HCC development and identifies ZBED3 as a key downstream effector in this process.