The aim of this study has been to identify transcription factors involved in transcriptional regulation of the human SOX18 gene expression. Structural analysis revealed that the SOX18 promoter lacks a TATA box, but is CG-rich containing many putative binding sites for transcription factors that can bind and act through GC-boxes. Alignment analysis of promoter regions between human and mouse revealed conserved putative binding sites for transcription factors NF-Y and Sp-family members. Mithramycin A treatment led to increased SOX18 expression in vivo raising the possibility that the GC-rich sequence of the human SOX18 promoter might be occupied by transcription factor(s) that acts as repressor(s). Using in vitro binding assays we have demonstrated that transcription factors Sp3, ZBP-89 and NF-Y are capable of binding to the SOX18 promoter region spanning the sequence -200 to -162 relative to ATG and that formation of complexes could be efficiently reduced by mithramycin A. Furthermore, co-transfection experiments revealed that over-expression of Sp3 and ZBP-89 down-regulate, while over-expression of NF-Y up-regulates SOX18 promoter activity in HeLa cells. The involvement of these transcription factors in the regulation of SOX18 expression in HeLa cells was further confirmed in vivo by Western blot analyses. In this paper, for the first time, we have demonstrated that Sp3, ZBP-89 and NF-Y are involved in transcriptional regulation of the human SOX18 gene expression. Presented data provide the initial information about transcriptional regulation that will help in better understanding of molecular mechanisms involved in regulation of SOX18 gene expression.