The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. In order to better understand the regulation of BTB dynamics, we report findings herein to understand the role of rpS6 (ribosomal protein S6), a downstream signaling protein of mTORC1 (mammalian target of rapamycin complex 1), in promoting BTB disruption in the testis in vivo, making the barrier "leaky". Overexpression of rpS6-WT (wild type, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and p-rpS6-MT (a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo) versus control (empty pCI-neo vector) was performed by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI® reagent. p-rpS6-MT was found to induce BTB disruption better than rpS6-WT (and no effects in empty vector control) based on an in vivo functional BTB integrity assay, leading to defects in spermatogenesis, including loss of spermatid polarity, failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, the mTORC1/rpS6 is a spermatogenesis and BTB regulator by modulating the organization of the actin- and MT-based cytoskeletons.