A local axis connects the apical ectoplasmic specialization (apical ES) at the Sertoli-spermatid interface, the basal ES at the blood-testis barrier (BTB), and the basement membrane across the seminiferous epithelium functionally in rat testes. As such, cellular events that take place across the epithelium such as spermiation and BTB remodeling that occur at the apical ES and the basal ES, respectively, at stage VIII of the cycle are coordinated. Herein, laminin α2, a structural component of the basement membrane, was found to regulate BTB dynamics. Sertoli cells were cultured in vitro to allow the establishment of a tight junction (TJ)-barrier that mimicked the BTB in vivo. Knockdown of laminin α2 by transfecting Sertoli cells with laminin α2-specific shRNA versus the non-targeting negative control was shown to perturb the Sertoli cell TJ-barrier, illustrating laminin α2 was involved in regulating BTB dynamics. This regulatory effect was mediated through mTORC1 signaling since the two mTORC1 downstream signaling molecules rpS6 and Akt1/2 was activated and inactivated, respectively, consistent with earlier findings that mTORC1 is involved in promoting BTB remodeling. Also, laminin α2 knockdown also induced F-actin and microtubule (MT) disorganization through changes in the spatial expression of F-actin regulators Arp3 and Eps8 vs. EB1 (a MT plus-end tracking protein, +TIP). These laminin α2 knockdown-mediated effects on F-actin and MT organization was blocked by exposing Sertoli cells to rapamycin, an inhibitor of mTORC1 signaling, and also SC79, an activator of Akt. In summary, laminin α2-mediated regulation on Sertoli cell BTB dynamics is through mTORC1 signaling.