Medulloblastoma, originating in the cerebellum, is the most common malignant brain tumor in children accounting for 15-25% of pediatric brain cancers. While drug therapies are available, none are consistently effective at overcoming resistance and killing cancer stem cells (CSCs). As a result, there is an urgent need for a new therapeutic approach. This study investigates the efficacy of a newly developed compound, VNPP433-3β, and its molecular mechanism on medulloblastoma cell lines and medullospheroids derived from medulloblastoma cell lines. DAOY and D341 metastatic cell lines were used as in vitro models, and medullospheroids were used as an ex vivo model. The cells were cultured and treated with specific media containing growth factor, and their viability was assessed using the MTT assay to determine the IC50 value at 24 h, 48 h, and 72 h. Morphological assessment was conducted using an inverted microscope, and inhibitory properties were determined in a clonogenic assay. The MTT assays revealed a significant reduction in cell viability in a time and dosedependent manner. Furthermore, treatment of cells with 1 μM of VNPP433-3β resulted in significantly fewer colonies and medullospheroids than untreated cells. The mechanism of cell death was examined using DAPI staining, which showed nuclear fragmentation and chromatin condensation, indicating cell death characteristic of apoptosis. Western blot analysis revealed that VNPP433-3β downregulated the oncoprotein FoxM1. Additionally, phospho-β-catenin expression, associated with spheroid formation, was downregulated compared with untreated cells. These findings suggest that VNPP433-3β has the potential to be a therapeutic candidate for the treatment of medulloblastomas