NSUN5, a 28S rRNA methyltransferase, regulates RNA metabolism by modifying 5-methylcytosine (m5C) to 5-hydroxymethylcytosine (5hmC) on chromatin-associated RNA (caRNA), which is subsequently degraded by RBFOX2. This process suppresses β-catenin and CD47, enhancing tumor-associated macrophage (TAM) phagocytosis and glioma elimination. NSUN5's methyltransferase activity, mediated by cysteine 359, is crucial for this function, and its localization in the nucleolus does not affect it. NSUN5 also recruits TET2 to chromatin, where TET2 oxidizes m5C to 5hmC, which is recognized and degraded by RBFOX2. This pathway is epigenetically regulated by DNA methylation and is negatively correlated with IDH1-R132H mutations in glioma patients. Pharmacological inhibition of DNA methylation or IDH1-R132H mutant and CD47/SIRPα signaling synergistically enhances TAM-based phagocytosis and glioma elimination in vivo. NSUN5 targeting represents a potential strategy for glioma immune therapy. The study reveals a general mechanism by which NSUN5/TET2/RBFOX2 signaling regulates RNA metabolism and highlights the therapeutic potential of NSUN5 in glioma immune therapy.NSUN5, a 28S rRNA methyltransferase, regulates RNA metabolism by modifying 5-methylcytosine (m5C) to 5-hydroxymethylcytosine (5hmC) on chromatin-associated RNA (caRNA), which is subsequently degraded by RBFOX2. This process suppresses β-catenin and CD47, enhancing tumor-associated macrophage (TAM) phagocytosis and glioma elimination. NSUN5's methyltransferase activity, mediated by cysteine 359, is crucial for this function, and its localization in the nucleolus does not affect it. NSUN5 also recruits TET2 to chromatin, where TET2 oxidizes m5C to 5hmC, which is recognized and degraded by RBFOX2. This pathway is epigenetically regulated by DNA methylation and is negatively correlated with IDH1-R132H mutations in glioma patients. Pharmacological inhibition of DNA methylation or IDH1-R132H mutant and CD47/SIRPα signaling synergistically enhances TAM-based phagocytosis and glioma elimination in vivo. NSUN5 targeting represents a potential strategy for glioma immune therapy. The study reveals a general mechanism by which NSUN5/TET2/RBFOX2 signaling regulates RNA metabolism and highlights the therapeutic potential of NSUN5 in glioma immune therapy.