BACKGROUND: NSUN5, also known as NOP2/Sun domain 5, is a pivotal RNA methyltransferase that catalyzes the formation of 5-methylcytosine (m5C). Cuproptosis, induced by elevated copper concentrations, is under investigation as a potential therapeutic strategy for cancer treatment. Despite this, the specific roles and the molecular mechanisms underlying Cuproptosis and NSUN5-mediated m5C modification in cholangiocarcinoma (CCA) remain to be fully elucidated. METHODS: Human tissue samples were collected to assess the expression levels of NSUN5 in CCA. In vitro functional assays were conducted to evaluate the biological function of NSUN5. The functional mechanism of NSUN5 on glutaminase (GLS) was investigated using RNA pull-down, RNA immunoprecipitation, molecular docking, and RNA stability assays. RESULTS: This study identified an upregulation of NSUN5 in CCA tissues. The knockdown of NSUN5 diminished the proliferation, migration, and invasion capabilities of CCA cells in vitro. In contrast, the overexpression of NSUN5 enhanced the growth and metastasis of CCA cells. Additionally, an increased copper content was detected in CCA tissues, which correlated with aggressive clinical features. CCA cells exhibited resistance to cuproptosis by upregulating GLS expression. Functionally, NSUN5 was found to positively modulate GLS expression. The NSUN5-mediated m5C modification at site 137 C on the GLS mRNA sequence stabilizes the GLS mRNA, leading to an accumulation of GLS within cells. CONCLUSIONS: Our findings highlight the critical role of NSUN5 in CCA progression through m5C-dependent stabilization of the GLS transcript, suggesting a potential targeted therapeutic strategy for CCA.