Osteosarcoma (OS) is a highly malignant bone tumor primarily affecting children and adolescents, with a significant portion of patients developing metastasis, leading to poor prognosis. Recent studies have identified long noncoding RNAs (lncRNAs) as critical regulators in cancer progression. Among these, LINC01082 has shown tumor-suppressive roles in various cancers, but its function and regulatory mechanisms in OS remain unclear.We investigated the expression patterns and biological functions of LINC01082 in OS tissues and cell lines using RT-qPCR, western blotting, and cell viability assays. The regulatory impact of 5-methylcytosine (m5C) RNA modification on LINC01082 stability was assessed through MeRIP-qPCR, RNA immunoprecipitation (RIP), and CRISPR/dCas13b-NSUN2-mediated m5C targeting. We further explored the interaction between LINC01082, miR-543, and TNRC6A within RNA-induced silencing complexes (RISCs) using luciferase reporter assays, RNA pull-down, and functional assays.LINC01082 was significantly downregulated in OS tissues and cell lines, with lower expression levels correlating with poorer patient survival. M5C modification, mediated by NSUN2, stabilized LINC01082 through its interaction with the m5C reader protein YBX1. CRISPR/dCas13b-NSUN2-mediated m5C targeting increased LINC01082 expression, resulting in reduced OS cell proliferation and migration, and increased apoptosis. Further, LINC01082 was found to positively regulate TNRC6A expression, with miR-543 modulating this interaction within RISCs. Inhibition of TNRC6A reversed the tumor-suppressive effects of LINC01082 methylation, highlighting the functional significance of the LINC01082-TNRC6A axis in OS.Our study highlights a novel m5C-dependent regulatory mechanism of LINC01082, and demonstrates the potential of CRISPR/dCas13b-NSUN2-mediated m5C editing to functionally modulate this lncRNA and suppress osteosarcoma progression.