Background <p>Neuroblastoma (NB) is a common pediatric malignancy originating from neural crest progenitor cells. While O-GlcNAcylation is known to regulate cancer cell metabolism and behavior, its specific role and prognostic value in neuroblastoma remain poorly understood. This study aims to elucidate the clinical significance and molecular mechanisms of O-GlcNAcylation in NB.</p> Methods <p>We analyzed O-GlcNAcylated protein expression in 158 human NB tumor samples using immunohistochemistry (IHC) and correlated the findings with clinicopathological parameters and survival outcomes. The therapeutic potential of enhancing O-GlcNAcylation via the OGA inhibitor Thiamet G was evaluated in MYCN-amplified NB cell lines and the <i>Th-MYCN</i> transgenic mouse model. Molecular mechanisms governing MYCN stability were investigated using Western blotting, immunoprecipitation, and functional assays.</p> Results <p>High levels of O-GlcNAcylated proteins were significantly associated with differentiated histology and early clinical stages. Survival analysis identified high O-GlcNAc expression as an independent prognostic factor for favorable outcomes. In vitro and in vivo experiments demonstrated that Thiamet G treatment effectively suppressed tumor growth and invasion while promoting neuronal differentiation. Mechanistically, Thiamet G-induced O-GlcNAc accumulation reduced inhibitory phosphorylation of GSK3β at Ser9, thereby activating GSK3β. This activation promoted the phosphorylation of MYCN at Thr58, accelerating its degradation via the ubiquitin-proteasome pathway.</p> Conclusion <p>Our findings demonstrate that high O-GlcNAcylated protein levels predict a favorable prognosis in neuroblastoma. Pharmacological inhibition of OGA with Thiamet G destabilizes the MYCN oncoprotein via the GSK3β-proteasome axis, suppressing tumorigenesis and inducing differentiation. This suggests that modulating O-GlcNAc levels represents a promising therapeutic strategy for MYCN-driven neuroblastoma.</p>