The serotonin transporter (5-HTT) gene contains a variable number tandem repeat (VNTR) domain within intron 2 that is often associated with a number of neurological conditions, including affective disorders. The implications of this polymorphism are not yet understood, however, we have previously demonstrated that the 5-HTT VNTR is a transcriptional regulatory domain, and the allelic variation supports differential reporter gene expression in vivo and in vitro. The aim of this study was to identify transcription factors responsible for the regulation of this VNTR. Using a yeast one-hybrid screen, we found the transcription factor Y box binding protein 1 (YB-1) interacts with the 5-HTT VNTR. Consistent with this, we demonstrate in a reporter gene assay that the polymorphic VNTR domains differentially respond to exogenous YB-1 and that YB-1 will bind to the VNTR in vitro in a sequence-specific manner. Interestingly, the transcription factor CCTC-binding factor (CTCF), previously shown to interact with YB-1, interferes with the ability of the VNTR to support YB-1-directed reporter gene expression. In addition, CTCF blocks the binding of YB-1 to its DNA recognition sequences in vitro, thus providing a possible mechanism of regulation of YB-1 activation of the VNTR by CTCF. Therefore, we have identified YB-1 and CTCF as transcription factors responsible, at least in part, for modulation of VNTR function as a transcriptional regulatory domain. Our data suggest a novel mechanism that explains, in part, the ability of the distinct VNTR copy numbers to support differential reporter gene expression based on YB-1 binding sites.