Neuroblastoma is one of the most common childhood solid tumors. 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, which mediates de novo DNA methylation, reportedly induces death in several types of human cancer cells. To study the antitumor activity of nanaomycin A against neuroblastoma cell lines and its mechanism. The anti-tumor effect of nanaomycin A on neuroblastoma cell lines was evaluated based on cell viability, DNA methylation levels, apoptosis-related protein expression, and neuronal-associated mRNA expression. Nanaomycin A decreased genomic DNA methylation levels and induced apoptosis in human neuroblastoma cells. Nanaomycin A also upregulated the expression of mRNAs for several genes related to neuronal maturation. Nanaomycin A is an effective therapeutic candidate for treating neuroblastoma. Our findings also suggest that the inhibition of DNA methylation is a promising anti-tumor therapy strategy for neuroblastoma.