Ki-67 plays a crucial role in cell proliferation as well as maintenance or regulation of cell division. The mechanism governing the Ki-67 gene expression remains unknown. Thus, we cloned the core promoter of the human Ki-67 gene and further investigated its transcriptional regulation. The putative Sp1 binding sites were confirmed by electrophoretic mobility shift assay together with an anti-Sp1 antibody-mediated supershift assay. Deletion mutagenesis and firefly luciferase reporter gene assay demonstrated the essential contribution of Sp1 on transcriptional activation of the Ki-67 gene. In this study, we first confirm that there are three Sp1 binding sites in the Ki-67 core promoter. Two Sp1 sites (one at position -159 to -145 nt and the other at position -14 to +12 nt) are mainly involved in transcriptional regulation of the Ki-67 gene. Overexpression of Sp1 can enhance the Ki-67 promoter activity. However, down-regulation of Sp1 expression using siRNA-Sp1 and mithramycin effectively inhibits the Ki-67 gene transcription. Our results suggest that Sp1 is essential for basal promoter activity of the human Ki-67 gene. Inhibition of the Ki-67 transcriptional activity through abolishment of Sp1 may provide the useful prospect for gene therapy.