Changes in epigenetic processes such as histone acetylation are proposed as key events influencing cancer cell function and the initiation and progression of pediatric brain tumors. Valproic acid (VPA) is an antiepileptic drug that acts partially by inhibiting histone deacetylases (HDACs) and could be repurposed as an epigenetic anticancer therapy. Here, we show that VPA reduced medulloblastoma (MB) cell viability and led to cell cycle arrest. These effects were accompanied by enhanced H3K9 histone acetylation (H3K9ac) and decreased expression of the oncogene. VPA impaired the expansion of MB neurospheres enriched in stemness markers and reduced while increasing expression in these neurospheres. In addition, VPA induced morphological changes consistent with neuronal differentiation and the increased expression of differentiation marker genes and . The expression of stemness genes , , and was differentially affected by VPA in MB cells with different status. VPA increased H3K9 occupancy of the promoter region of . Among the genes regulated by VPA, the stemness regulators and showed an association with patient survival in specific MB subgroups. Our results indicate that VPA may exert antitumor effects in MB by influencing histone acetylation, which may result in the modulation of stemness, neuronal differentiation, and the expression of genes associated with patient prognosis in specific molecular subgroups. Importantly, the actions of VPA in MB cells and neurospheres include a reduction in the expression of and an increase in .