Neuroblastoma, a prevalent pediatric solid tumor, is characterized by substantial genetic, morphological, and clinical heterogeneity, which poses challenges to the efficacy of existing therapeutic interventions. Previous studies have demonstrated that the histone deacetylase (HDAC) inhibitor MS275 can impede the growth of neuroblastoma cells. However, the precise mechanisms underlying this inhibitory effect remain inadequately understood. To assess the impact of MS275 on neuroblastoma cell proliferation, a series of assays, including CCK8, colony formation assay, EdU assay, cell cycle assay, and apoptosis assays, were conducted. Furthermore, the qRT-PCR, western blotting, luciferase reporter assay, and chromatin immunoprecipitation (ChIP) assays were utilized to elucidate the regulatory relationship between MS275 and PROX1 expression. To further ascertain the role of PROX1 in MS275-mediated tumor suppression, in silico analyses were complemented by the establishment of PROX1 knockdown SH-SY5Y cell lines and xenograft models. The cell functional assays revealed that MS275 exerts an inhibitory effect on neuroblastoma cell proliferation by inducing G1/S cell cycle arrest, apoptosis, and autophagy in a dose-dependent manner. Notably, the reduction of PROX1 expression was observed to partially reverse the inhibitory effects of MS275 on neuroblastoma cells, affecting cell cycle progression, apoptosis, autophagy-related factors, and tumor growth. Mechanistically, MS275 was found to significantly enhance and modify the transcription of PROX1 through the induction of H3K27 acetylation. In summary, these findings highlight the crucial role of PROX1 in MS275-mediated tumor suppression and unveil a novel regulatory mechanism involving PROX1, suggesting its potential as a promising therapeutic target for neuroblastoma.