We identified the promoter region of the human GD3 synthase (hST8Sia I) gene to elucidate the mechanism underlying the regulation of hST8Sia I expression in human glioblastoma cells. The 5′-rapid amplification of cDNA end using mRNA prepared from U-87MG cells revealed the presence of transcription start site of hST8Sia I gene, and the 5′-terminal analysis of its product showed that transcription started from 648 nucleotides upstream of the translational initiation site. Functional analysis of the 5′-flanking region of the hST8Sia I gene by transient expression method revealed that the region from −638 to −498 is important for transcriptional activity of the hST8Sia I gene in U-87MG and T98G cells. This region lacks apparent TATA and CAAT boxes, but contains putative binding sites for transcription factors AREB6 and Elk-1. Site-directed mutagenesis and transient transfection assays demonstrated that both AREB6 and Elk-1 elements in this region were required for the promoter activity in U-87MG and T98G cells. These results indicated that both AREB6 and Elk-1 might play an essential role in the transcriptional activity of hST8Sia I gene essential for GD3 synthesis in human glioblastoma cells.