Transcriptional regulation mechanisms have not been clearly illuminated for NGX6 gene, which is a candidate of tumor suppressor gene in colorectal cancer. pGL3/Enhancer/1126 vector, a recombinant reporter gene vectors of the transcription regulatory region of NGX6 gene, was constructed based on bioinformatic techniques and identified by luciferase assay system. No canonical TATA boxes, but several CAAT and GC boxes were observed in the transcription regulatory region by the online analysis programs PromoterInspector program, FistEF, CpGplot and MatInspector Professional. Transcriptional factor Sp1 was validated to bind to NGX6 promoter by electrophoretic mobility shift assay (EMSA). Inhibition of the Sp1 binding to NGX6 promoter by mithramycin A significantly reduced the promoter activity. The endogenous expression of NGX6 in mRNA level was down-regulated by mithramycin A and blocking with Sp1.