Objective To investigate the regulation difference of Sp1 binding sites ( - 170 ～- 145 nt) in the human Ki-67 gene promoter which contains two coupled Spl binding sites and further illuminate the potential cause of Ki-67 high expression in the tumor cells.Methods Site-directed mutagenesis and firefly luciferase reporter gene assay were used to detect the contribution of these Sp1 binding sites mutations Mut A ( - 170/- 160 nt) and Mut B ( - 159/- 145 nt) on transcriptional activation of the Ki-67 gene in Hela cells,OS-RC-2 cells and A549 cells,respectively.Results The transcriptional activity of the two mutant plasmids were decreased in these three cancer cells,and the decline of the transcriptional activity of mutant B is greater.In Hela cells,OS-RC-2 cells,A549 cells the relative dual luciferase reporter gene of mutant B exhibited transcriptional activities respectively equivalent to 36.9%,26.9% and 28.6% of plasmid pGLBK235.Conclusion Spl binding sites ( - 159/- 145 nt) in the human Ki-67 gene promoter is essential for basal transcription activation of the human Ki-67 gene.