The blood-testis barrier (BTB) is an important ultrastructure in the testis that supports meiosis and postmeiotic spermatid development since a delay in the establishment of a functional Sertoli cell barrier during postnatal development in rats or mice by 17–20 day postpartum (dpp) would lead to a delay of the first wave of meiosis. Furthermore, irreversible disruption of the BTB by toxicants also induces infertility in rodents. Herein, we summarize recent findings that BTB dynamics (i.e., disassembly, reassembly, and stabilization) are supported by the concerted efforts of the actin- and microtubule (MT)-based cytoskeletons. We focus on the role of two actin nucleation protein complexes, namely, the Arp2/3 (actin-related protein 2/3) complex and formin 1 (or the formin 1/spire 1 complex) known to induce actin nucleation, respectively, by conferring plasticity to actin cytoskeleton. We also focus on the MT plus (+)-end tracking protein (+TIP) EB1 (end-binding protein 1) which is known to confer MT stabilization. Furthermore, we discuss in particular how the interactions of these proteins modulate BTB dynamics during spermatogenesis. These findings also yield a novel hypothetical concept regarding the molecular mechanism that modulates BTB function.