The blood-testis barrier (BTB) in the testis is an important ultrastructure to support spermatogenesis. This blood-tissue barrier undergoes remodeling at late stage VII-early stage IX of the epithelial cycle to support the transport of preleptotene spermatocytes across the BTB to prepare for meiosis I/II at the apical compartment through a mechanism that remains to be delineated.. Studies have shown that NC1-peptide derived collagen α3 (IV) chain in the basement membrane, is a bioactive peptide that induces BTB remodeling. It also promotes the release of fully developed spermatids into the tubule lumen. Thus, this endogenously produced peptide coordinates these two cellular events across the epithelium. Using an NC1-peptide cDNA construct to transfect adult rat testes for its overexpression, NC1-peptide was found to effectively induce germ cell exfoliation, which was associated with a surge and activation of p-rpS6, the downstream signaling protein of mTORC1, and a concomitant down-regulation of p-FAK-Y407 in the testis. In order to define the functional relationship between p-rpS6 and p-FAK-Y407 signaling to confer NC1-peptide the ability to regulate testis function, a phosphomimetic (and thus constitutively active) mutant of p-FAK-Y407, p-FAK-Y407E-MT, was used for its co-transfection with NC1-peptide using Sertoli cells cultured in vitro that mimicked the BTB in vivo. Overexpression of p-FAK-Y407E-MT blocked the effects of NC1-peptide to perturb Sertoli cell BTB function by promoting F-actin and microtubule cytoskeleton function, and downregulated the NC1-peptide mediated induction of p-rpS6 activation. In brief, NC1-peptide is an important endogenously produced biomolecule to regulate BTB dynamics.