The expression of claudin-11, a key integral tight junction protein, is tightly regulated to ensure that the integrity of the seminiferous epithelium could be maintained during the translocation of spermatocytes at the blood-testis barrier at stages VIII-IX. In this study, we elucidate how the overlapping GATA/NF-Y motif within the core promoter of claudin-11 gene is modulated by differential binding of various transcription factors, resulting in dual transcriptional control. Using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay, we confirmed that GATA, nuclear factor YA (NF-YA), and cAMP response element-binding protein (CREB) form a complex in vivo and bind to the GATA/NF-Y region to promote claudin-11 gene transcription. Such gene activations were significantly reduced in the presence of siRNA specific to these transcription factors. GATA and CREB transactivation could be further modulated by the presence of Smad3 and Smad4 proteins. Binding of Smad proteins at the GATA/NF-Y motif could repress the GATA and CREB transactivation of claudin-11 gene. Such repression which required the recruitment and physical interactions of histone deacetylase 1 and its co-repressor, mSin3A with Smad proteins, was abolished by treatment with Trichostatin A, thus suggesting the involvement of histone deacetylation at the site of the promoter region. It is believed that cyclic changes in the ratio of positive regulators (GATA, NF-Y, and CREB) to negative regulators (Smads) in the seminiferous epithelium during the spermatogenic cycle might provide a precise control in claudin-11 gene transcription.