The mechanism of transcriptional silencing of ST6Gal1 in gliomas has not yet been elucidated. Multiple independent promoters govern the expression of the ST6Gal I gene. Here, we investigated whether epigenetic abnormalities involving DNA methylation affect ST6Gal1 expression. Transcript-specific qRT-PCR following exposure of glioma cell lines to 5-aza-2’-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, resulted in the re-expression of the normally quiescent ST6Gal1 mRNA driven exclusively by the P3 promoter sequence. The P3 promoter-specific transcription start site (TSS) was delineated by primer extension and core promoter sequences and associated functional transcription elements identified by deletion analysis utilizing chloramphenicol acetyltransferase reporter constructs. Minimal promoter activity was found to reside within the first 100 bp of the TSS and maximal activity was controlled by functional AP2 binding sites residing between 400 and 500 bp upstream of the initiation site. As altered AP2 binding was not directly associated with AP2 availability, these analyses demonstrate that ST6Gal1 transcription is regulated by DNA methylation within core promoter regions, ultimately by determining critical transcription factor accessibility within these regions. Transcriptional reactivation of ST6Gal1 expression by 5-aza-dC resulted in increased cell surface α2,6 sialoglycoconjugate expression, increased α2,6 sialylation of β1 integrin, and decreased adhesion to fibronectin substrate: functional correlates of decreased invasivity. The effects of global hypomethylation are not glycome-wide. Focused glycotranscriptomic analyses of three invasive glioma cell lines following 5-aza-dC treatment demonstrated the modulation of select glycogene transcripts. Taken together, these results demonstrate that epigenetic modulation of ST6Gal1 expression plays a key role in the glioma phenotype in vitro and that that therapeutic approaches targeting elements of the epigenetic machinery for the treatment of human glioblastoma are warranted.