In the mammalian testis, junctional adhesion molecule-B (JAM-B) is found at the blood-testis barrier between Sertoli cells and the apical ectoplasmic specializations between Sertoli and germ cells. The expression of JAM-B is tightly regulated to allow the transit of developing germ cells across the blood-testis barrier and the timely release of mature spermatids at stage VIII. In this study, the basal transcription of JAM-B in the mouse Sertoli cell line, MSC-1 cells, was examined. We found that the constitutive expression of JAM-B is carried out by the binding of specificity proteins (Sps), ETS domain transcription factor Elk-1 (Elk1), neuron-restrictive silencer factor (NRSF), and E2F transcription factor 3 (E2F3) to various cis-acting elements including TG interacting factor (TGIF), Elk-1, NRSF, and proximal Sp1 (pSp1) + E2F binding motifs. We also investigated the effects of two cytokines IL-1α and TGF-β2 on JAM-B expression. IL-1α promotes JAM-B expression by facilitating the binding of Elk-1 to TGIF and pSp1 + E2F motifs in a p38-dependent manner, which leads to an additive effect on Sp1- and NRSF-mediated JAM-B transactivation. TGF-β2 inhibits JAM-B transcription via the activation of mothers against decapentaplegic (Smad) proteins and activated Smads compete with specificity proteins (Sp1 and Sp3) for the TGIF motif, resulting in JAM-B repression. IL-1α and Smad3 expression have been reported to be stage specific. IL-1α is absent in the seminferous epithelium at stages VII–VIII, whereas a high level of nuclear Smad3 level is found at the same stages. This study shows for the first time that IL-1α and TGF-β2 regulate JAM-B expression in an opposite manner, and in vitro data obtained herein provide some clues on how junctions are regulated in the testis.