Neuroblastoma is an often fatal pediatric cancer arising from precursor cells of the sympathetic nervous system. 13-Cis retinoic acid is included in the treatment regimen for patients with high-risk disease, and a similar derivative, all-trans-retinoic acid (ATRA), causes neuroblastoma cell lines to undergo differentiation. The molecular signaling pathways involved with ATRA-induced differentiation are complex, and the role that DNA methylation changes might play are unknown. The purpose of this study was to evaluate the genome-wide effects of ATRA on DNA methylation using methylated DNA immunoprecipitation applied to microarrays representing all known promoter and CpG islands. Four hundred and two gene promoters became demethylated, whereas 88 were hypermethylated post-ATRA. mRNA expression microarrays revealed that 82 of the demethylated genes were overexpressed by >2-fold, whereas 13 of the hypermethylated genes were underexpressed. Gene ontology analysis indicated that demethylated and re-expressed genes were enriched for signal transduction pathways, including NOS1, which is required for neural cell differentiation. As a potential mechanism for the DNA methylation changes, we show the downregulation of methyltransferases, DNMT1 and DNMT3B, along with the upregulation of endogenous microRNAs targeting them. Ectopic overexpression of miR-152, targeting DNMT1, also negatively affected cell invasiveness and anchorage-independent growth, contributing in part to the differentiated phenotype. We conclude that functionally important, miRNA-mediated DNA demethylation changes contribute to the process of ATRA-induced differentiation resulting in the activation of NOS1, a critical determinant of neural cell differentiation. Our findings illustrate the plasticity and dynamic nature of the epigenome during cancer cell differentiation.