Neuroblastoma is one of the most common childhood solid tumors. Despite intensive multidisciplinary treatment, the current 5-year overall survival rate for children diagnosed at an advanced stage is less than 50%, with a poor prognosis. Chemotherapeutic agents commonly used to treat high-risk neuroblastomas (e.g., those involving MYCN amplifications), such as cyclophosphamide, cisplatin and so on, are toxic to proliferating cells, including normal cells. Thus, new therapeutic strategies are needed to improve the prognosis of children with neuroblastoma. DNA methylation is an epigenetic modification that suppresses gene expression. Because tumor suppressor genes are often hypermethylated in cancers, DNA methylation has emerged as a target for cancer therapeutics. Nanaomycin A, an inhibitor of DNA methyltransferase 3B (DNMT3B) which mediates de novo DNA methylation and is rarely expressed in normal cells, reportedly induces death in several types of human cancer cells. In the present study, nanaomycin A decreased genomic DNA methylation levels and induced apoptosis in human neuroblastoma cells. These results suggest that nanaomycin A is an effective candidate therapeutic for treating neuroblastoma. Our findings also suggest that the inhibition of DNA methylation is a promising anti-tumor therapy strategy for neuroblastoma.