SRY-related HMG-box genes (Sox genes) constitute a large family of developmentally regulated genes involved in the decision of cell fates during development and implicated in the control of diverse developmental processes. Sox3, an X-linked member of the family, is expressed in the central nervous system (CNS) from the earliest stages of development. It is considered to be one of the earliest neural markers in vertebrates playing the role in specifying neuronal fate. The aim of this study has been to determine and characterize the promoter of the human SOX3 gene and to elucidate molecular mechanisms underlying the regulation of its expression. In this study, we have isolated and performed the first characterization of the human SOX3 promoter. We have identified the transcription start point (tsp) and carried out the structural and functional analysis of the regulatory region responsible for SOX3 expression in NT2/D1 cell line. Using promoter-reporter constructs, we have determined the minimal SOX3 promoter region that confers the basal promoter activity, as well as two regulatory elements which have positive effects on the promoter activity. We have investigated in detail the functional properties of three conserved motifs within the core promoter sequence that bind transcription factors specificity protein 1 (Sp1), upstream stimulatory factor (USF) and nuclear factor Y (NF-Y). By mutational analysis, we have shown that all three sites are of functional relevance for constitutive SOX3 expression in NT2/D1 cells. We have also shown that, besides the TATA motif, at least one other essential regulatory element is required for the basal transcription of the human SOX3. Taken together, data presented in this paper suggest that transcription factors such as Sp1, USF and NF-Y could function as key regulators for the basal activation of the human SOX3 gene.