Spermatogenesis takes place in the epithelium of the seminiferous tubules of the testes, producing millions of spermatozoa per day in an adult male in rodents and humans. Thus, multiple cellular events that are regulated by an array of signaling molecules and pathways are tightly coordinated to support spermatogenesis. Here, we report findings of a local regulatory axis between the basement membrane (BM), the blood-testis barrier (BTB), and the apical ectoplasmic specialization (apical ES; a testis-specific, actin-rich adherens junction at the Sertoli cell–spermatid interface) to coordinate cellular events across the seminiferous epithelium during the epithelial cycle. In short, a biologically active fragment, noncollagenous 1 (NC1) domain that is derived from collagen chains in the BM was found to modulate cell junction dynamics at the BTB and apical ES. NC1 domain from the collagen α3 (IV) chain was cloned into a mammalian expression vector, pCI-neo, with and without a collagen signal peptide. We also prepared a specific Ab against the purified recombinant NC1 domain peptide. These reagents were used to examine whether overexpression of NC1 domain with high transfection efficacy would perturb spermatogenesis, in particular, spermatid adhesion (i.e., inducing apical ES degeneration) and BTB function (i.e., basal ES and tight junction disruption, making the barrier leaky), in the testis in vivo. We report our findings that NC1 domain derived from collagen α3 (IV) chain—a major structural component of the BM—was capable of inducing BTB remodeling, making the BTB leaky in studies in vivo. Furthermore, NC1 domain peptide was transported across the epithelium via a microtubule-dependent mechanism and is capable of inducing apical ES degeneration, which leads to germ cell exfoliation from the seminiferous epithelium. Of more importance, we show that NC1 domain peptide exerted its regulatory effect by disorganizing actin microfilaments and microtubules in Sertoli cells so that they failed to support cell adhesion and transport of germ cells and organelles (e.g., residual bodies, phagosomes) across the seminiferous epithelium. This local regulatory axis between the BM, BTB, and the apical ES thus coordinates cellular events that take place across the seminiferous epithelium during the epithelial cycle of spermatogenesis.