SOX3 is a member of the Sox gene family implicated in brain formation and cognitive function. It is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. Recently, we have established the first link between TALE (three-amino-acid loop extension) proteins, PBX1 (pre-B-cell leukemia homeobox 1) and MEIS1 (myeloid ecotropic viral integration site 1 homologue), and the expression of the human SOX3 gene. Here we present the evidence that TGIF (TG-interacting factor) is an additional TALE superfamily member involved in the regulation of human SOX3 gene expression in NT2/D1 cells by direct interaction with the consensus binding site that is conserved in primate orthologue promoters. Functional analysis demonstrated that mutation of the TGIF binding site resulted in the activation of SOX3 promoter. TGIF overexpression downregulates SOX3 promoter activity and decreases endogenous SOX3 protein expression in both uninduced and retinoic acid (RA)-induced NT2/D1 cells. Up to now, this is the first transcription factor identified as a negative regulator of SOX3 gene expression. The obtained results further underscore the significance of TALE proteins as important transcriptional regulators of SOX3 gene expression.