MicroRNAs (miRNAs) are important post-transcriptional regulators of various biological processes. Although our knowledge of miRNA expression and regulation has been increased considerably in recent years, the regulatory elements for miRNA gene expression (especially for intergenic miRNAs) are not fully understood. In this study, we identified differentially methylated regions (DMRs) within 1000 bp upstream from the start site of intergenic miRNAs in human neuroglioma cells using microarrays. Then we identified a unique sequence pattern, C[N](6)CT, within the DMRs using motif analysis. Interestingly, treatment of cells with a methyl transferase inhibitor (5-aza-2-deoxycytidine, DAC) significantly increased expression of miRNA genes with a high frequency of the C[N](6)CT motif in DMRs. Statistical analysis showed that the frequency of the C[N](6)CT motif in DMRs is highly correlated with intergenic miRNA gene expression, suggesting that C[N](6)CT motifs associated with DNA methylation regions play a role as regulatory elements for intergenic miRNA gene expression. (C) 2012 Elsevier Inc. All rights reserved.