Regulation of tight junction protein expressions in Sertoli cells is important for germ cell translocation across the blood-testis-barrier (BTB) during spermatogenesis. In this study, a novel tight junction transmembrane protein, CLMP, found expressed in mouse testis was shown to localize at the BTB along with the tight junction marker ZO-1. By the use of transient transfection assay performed in a mouse Sertoli cell–cell line, TM4 cells, we showed that the minimal CLMP promoter was located between nucleotides −550 and −288 relative to the translation start site. Site-directed mutagenic studies showed that three motifs, namely GATA, KLF4, and SRY, within this region functionally co-operated with one another to regulate CLMP gene transcription. Using specific antibodies in EMSA analysis, a ternary protein complex GATA-1/GATA-6/KLF4 was detected at all the three motifs, suggesting that a looping mechanism might involve in regulating CLMP gene transcription. Interestingly, the ubiquitously expressed transcription factors, Sp1 and Sp3, were also found in this ternary complex over the KLF4 motif. Overexpression of KLF4 significantly increased the promoter activity whilst overexpression of Sp1 or Sp3 exerted an opposite effect. In particular, co-transfection studies showed that Sp1 could significantly abolish the KLF4-induced transactivation of the CLMP gene, suggesting that KLF4 and Sp1 might compete for the same binding site on the CLMP promoter. Taken together, this differential interaction of the transcription factors, GATA-1, GATA-6, KLF4, Sp1, and Sp3, in CLMP gene expression might provide a precise machinery in regulating Sertoli cell tight junction dynamics. J. Cell. Physiol. 214: 334–344, 2008. © 2007 Wiley-Liss, Inc.