The NXF (nuclear export factor) family members are implicated in the transport of mRNA from the nucleus to the cytoplasm. Recently, some members of the NXF family have been reported to play divergent functional roles, such as post-transcriptional regulation, translational control, regulation of mRNA stability and trafficking. However, little is known about the roles of NXF3 in spermatogenesis. In the present study, we found that mouse NXF3, specifically expressed in principal cells in segment II of the caput epididymis, as well as Sertoli cells in the mouse testis, was required to mediate TGF-β (transforming growth factor β)-induced down-regulation of Tgfb3/TGF-β3 mRNA expression and protein secretion in Sertoli cells. In addition, NXF3 was also involved in TGF-β-induced transcriptional regulation of other genes associated with Sertoli cell maturation and the restructuring of the Sertoli cell BTB (blood-testis barrier), such as Gata1 (GATA-binding protein 1), Wt1 (Wilms's tumour homologue 1), Cldn11 (claudin11) and Cdkn1a (cyclin-dependent kinase inhibitor 1A or p21(Cip1)). The transcriptional regulation of NXF3 was mediated through physical interaction with STRAP (serine/threonine kinase receptor-associated protein), where NXF3 inhibited the complex formation among Smad7, STRAP and activated type I TGF-β receptor. Taken together, our data provide mechanistic insights into the roles of NXF3 in TGF-β-mediated expression of Tgfb3 and other genes. NXF3 may be implicated in Sertoli cell maturation and the extensive restructuring of the Sertoli cell BTB.